Nucleotide sequences that code for torsin genes, torsin proteins, and methods of using the same to treat protein-aggregation

ABSTRACT

The invention relates to polynucleotides comprising polynucleotide sequences corresponding to the tor- 1 , tor- 2 , ooc- 5 , DYT 1 , and DYT 2  genes and parts thereof that encode polypeptide sequences and parts thereof possessing varying degrees of torsin activity, and methods of screening and amplifying polynucleotides encoding polypeptide sequences which encode polypeptides having varying degrees of TOR- 1 , TOR 2 , OOC- 5  TOR-A, and TOR-B activity. Further, the invention relates to methods of reducing protein aggregation, methods of treating diseases that are caused by protein aggregation, methods of screening potential protein-aggregation-reducing products, methods of screening potential therapeutics of diseases caused by protein aggregation, and pharmaceuticals, therapeutics, and kits comprising polynucleotide sequences corresponding to the tor- 1 , tor- 2 , ooc- 5 , DYT 1 , and DYT 2  genes and/or polypeptides having torsin activity.

BACKGROUND OF INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to polynucleotides comprisingpolynucleotide sequences corresponding to the tor-1, tor-2, ooc-5, DYT1, and DYT2 genes and parts thereof that encode polypeptide sequencesand parts thereof possessing varying degrees of torsin activity, andmethods of screening and amplifying polynucleotides encoding polypeptidesequences which encode polypeptides having varying degrees of TOR-1,TOR2, OOC-5 TOR-A, and TOR-B activity. Further, the invention relates tomethods of reducing protein aggregation, methods of treating diseasesthat are caused by protein aggregation, methods of screening potentialprotein-aggregation-reducing products, methods of screening potentialtherapeutics of diseases caused by protein aggregation, andpharmaceuticals, therapeutics, and kits comprising polynucleotidesequences corresponding to the tor-1, tor-2, ooc-5, DYT1, and DYT2 genesand/or polypeptides having torsin activity.

[0003] 2. Discussion of the Background

[0004] Neuronal damage may be caused by toxic, aggregation-proneproteins. Further, an enormous scope of neurodegenerative disorders ischaracterized by such neuronal damage. Therefore, theseneurodegenerative disorders are inevitably a result of proteinaggregation. Genes have been identified that code for such toxic,aggregation-prone proteins which cause these disorders. Further,mutations in such genes result in abnormal processing and accumulationof misfolded proteins. These misfolded proteins are known to result inneuronal damage such as neuronal inclusions and plaques. Therefore, theunderstanding of the cellular mechanisms and the identification of themolecular tools required for the reduction, inhibition, and ameliorationof such misfolded proteins is critical. Further, an understanding of theeffects of protein aggregation on neuronal survival will allow thedevelopment of rational, effective treatment for these disorders.

[0005] Neuronal disorders, including early-onset torsion dystonia arecharacterized by uncontrolled muscular spasms. Dystonia is set apart inthat the muscle spasms are repetitive and rhythmic (Bressman, S B. 1998.Dystonia. Current Opinion in Neurology. 11:363-372). The symptoms canrange in severity from a writer's cramp to being wheelchair bound.Early-onset torsion dystonia, also called primary dystonia, isdistinguished by strong familial ties and the absence of any neuraldegeneration, which is seen in the other movement disorders. This ismost severe form of the disease and is dominantly inherited with a lowpenetrance (30%-40%) (L. J. Ozelius, et al., Genomics 62, 377 (1999); L.J. Ozelius, et al., Nature Genetics 17, 40 (1997)). Therefore, dystoniais difficult to diagnose and pathologically define. Dystonia affectsmore than 300,000 people in North America and is more common thanHuntington's disease and muscular dystrophy. Treatment is very limitedbecause the disease is poorly understood and options include surgery orinjection of botulism toxin to control the muscle contractions.

[0006] The molecular basis for torsion dystonia remains unclear. Ozeliuset al. identified the causative gene, named TOR 1A (DYT1), and mapped itto human chromosome 9q34 (L. J. Ozelius, et al., Nature Genetics 17, 40(1997)). The TOR 1A gene produces a protein named TOR-A. The majority ofpatients with early onset torsion dystonia have a unique deletion of onecodon, which results in a loss of glutamic acid (GAG) residue at thecarboxy terminal of TOR-A. A misfunctional torsin protein is produced.Notably, this was the only change observed on the disease chromosome (L.J. Ozelius, et al., Genomics 62, 377 (1999); L. J. Ozelius, et al.,Nature Genetics 17, 40 (1997)). A recent paper described an additionaldeletion of 18 base pairs or 6 amino acids at the carboxy terminus. Thisis the first mutation identified beyond the GAG deletion (L. J. Ozelius,et al., Nature Genetics 17, 40 (1997)).

[0007] In the original paper identifying the TOR1A gene, a nematodetorsin-like protein was described, which has since been shown to encodethe ooc-5 gene (L. J. Ozelius, et al., Nature Genetics 17, 40 (1997); S.E. Basham, and L. E. Rose, Dev Biol 215 253 (1999)). The TOR-A proteinshares a distant similarity (25%-30%) to the AAA+/Hsp 100/Clp family ofproteins (chromosome (L. J. Ozelius, et al., Genomics 62, 377 (1999);Neuwald A F, Aravind L, Spouge J L, Koonin E V. 1999. AAA+: A class ofchaperone-like ATPases associated with the assembly, operation, anddisassembly of protein complexes. Genome Res 9: 27-43). Their tasks areas diverse as their similarities. For example, they perform chaperonefunctions, regulate protein signaling, and allow for the correctlocalization of the proteins. However, until the time of the presentinvention, the function of torsin proteins has not been elucidated andtheir activities are unknown.

SUMMARY OF THE INVENTION

[0008] The present invention relates to dystonia, dystonia genes,encoded proteins and mutations in dystonia genes that result in adystonia disorder. In particular, the invention provides isolatednucleic acid molecules coding for torsin proteins, preferably, TOR-2.

[0009] The invention further provides purified polypeptides comprisingamino acid sequences contained in torsin proteins.

[0010] The invention also provides nucleic acid probes for the specificdetection of the presence of and mutations in nucleic acids encodingtorsin proteins or polypeptides in a sample.

[0011] The invention further provides a method of detecting the presenceof mutations in a nucleic acid encoding a torsin protein in a sample.

[0012] The invention also provides a kit for detecting the presence ofmutations in a nucleic acid encoding a torsin protein in a sample.

[0013] The invention further provides a recombinant nucleic acidmolecule comprising, 5′ to 3′, a promoter effective to initiatetranscription in a host cell and the above-described isolated nucleicacid molecule.

[0014] The invention also provides a recombinant nucleic acid moleculecomprising a vector and the above-described isolated nucleic acidmolecule.

[0015] The invention further provides a method of screening for acompound that reduces, inhibits, ameliorates, or prevents proteinaggregation by comparing the amount of protein aggregation in thepresence of the compound to the amount of protein aggregation in theabsence of the compound. This method of screening is performed in thepresence of at least one torsin protein. The torsin protein may bemutated.

[0016] The invention further provides a recombinant nucleic acidmolecule comprising a sequence complimentary to an RNA sequence encodingan amino acid sequence corresponding to the above-described polypeptide.

[0017] The invention also provides a cell that contains theabove-described recombinant nucleic acid molecule.

[0018] The invention further provides a non-human organism that containsthe above-described recombinant nucleic acid molecule.

[0019] The invention also provides an antibody having binding affinityspecifically to a torsin protein or polypeptide.

[0020] The invention further provides a method of detecting a torsinprotein or polypeptide in an sample.

[0021] The invention also provides a method of measuring the amount of atorsin protein or polypeptide in a sample.

[0022] The invention further provides a method of detecting antibodieshaving binding affinity specifically to a torsin protein or polypeptide.

[0023] The invention further provides a diagnostic kit comprising afirst container means containing a conjugate comprising a bindingpartner of the monoclonal antibody and a label.

[0024] The invention also provides a hybridoma which produces theabove-described monoclonal antibody.

[0025] The invention further provides diagnostic methods for dystoniadisorders in humans, in particular, torsion dystonia. Preferably, amethod of diagnosing the presence or absence of dystonia; predicting thelikelihood of developing or a predisposition to develop dystonia in ahuman is provided herein. The dystonia disorder can be, for example,torsion dystonia. A biological sample obtained from a human can be usedin the diagnostic methods. The biological sample can be a bodily fluidsample such as blood, saliva, semen, vaginal secretion, cerebrospinaland amniotic bodily fluid sample. Alternatively or additionally, thebiological sample is a tissue sample such as a chorionic villus,neuronal, epithelial, muscular and connective tissue sample. In bothbodily fluid and tissue samples, nucleic acids are present in thesamples.

[0026] The dystonia gene can be the tor-1, tor-2, ooc-5, DYT1, and DYT2genes, and parts thereof (SEQ ID NOS: 1, 3, 5, 7, and 9). In oneembodiment the gene may be mutated, such as a deletion mutation.Alternatively the mutation can be a missense, or frame shift mutation.For example, if the mutation to be detected is a deletion mutation, thepresence or absence of three nucleotides in this region.

[0027] The invention also relates to methods of detecting the presenceor absence of dystonia disorder in a human wherein the dystonia disorderis characterized by one or more mutations in a dystonia gene.

[0028] Another aspect of the invention relates to methods of detectingthe presence or absence of a dystonia disorder, wherein the test samplefrom the human is evaluated by performing a polymerase chain reaction,hereinafter “PCR,” with oligonucleotide primers capable of amplifying adystonia gene. Following PCR amplification of a nucleic acid sample, theamplified nucleic acid fragments are separated and mutations in thetor-2 gene and alleles of the dystonia gene detected. For example, amutation in the tor-2 gene is indicative of the presence of the torsiondystonia, whereas the lack of a mutation is indicative of a negativediagnosis.

[0029] An additional aspect of the invention is a method of determiningthe presence or absence of a dystonia disorder in a human including thesteps of contacting a biological sample obtained from the human with anucleic acid probe to a dystonia gene; maintaining the biological sampleand the nucleic acid probe under conditions suitable for hybridization;detecting hybridization between the biological sample and the nucleicacid probe; and comparing the hybridization signal obtained from thehuman to a control sample which does or does not contain a dystoniadisorder. The hybridization is performed with a nucleic acid fragment ofa dystonia gene such as SEQ ID NOS: 1, 3, 5, 7, and 9. The nucleic acidprobe can be labeled (e. g., fluorescent, radioactive, enzymatic, biotinlabel).

[0030] The invention also encompasses methods for predicting whether ahuman is likely to be affected with a dystonia disorder, comprisingobtaining a biological sample from the human; contacting the biologicalsample with a nucleic acid probe; maintaining the biological sample andthe nucleic acid probe under conditions suitable for hybridization; anddetecting hybridization between the biological sample and the nucleicacid probe. In another embodiment the method further comprisesperforming PCR with oligonucleotide primers capable of amplifying adystonia gene (e.g., SEQ ID NOS: 1, 3, 5, 7, and 9); and detecting amutation in amplified DNA fragments of the dystonia gene, wherein themutation in the dystonia gene is indicative of the presence or absenceof the torsion dystonia. The hybridization can detect, for example, adeletion in nucleotides indicative of a positive diagnosis; or thepresence of nucleotides indicative of a negative diagnosis.

[0031] The invention further provides for methods of determining thepresence or absence of a dystonia disorder in a human comprisingobtaining a biological sample from the human; and assessing the level ofa dystonia protein in the biological sample comprising bodily fluids,tissues or both from the human. The levels or concentrations of thedystonia protein are determined by contacting the sample with at leastone antibody specific to a dystonia protein, and detecting the levels ofthe dystonia protein. An alteration in the dystonia protein levels isindicative of a diagnosis. The antibody used in the method can be apolyclonal antibody or a monoclonal antibody and can be detectablylabeled (e. g., fluorescence, biotin, colloidal gold, enzymatic). Inanother embodiment the method of assessing the level or concentration ofthe dystonia protein further comprises contacting the sample with asecond antibody specific to the dystonia protein or a complex between anantibody and the dystonia protein.

[0032] The present invention also provides for a kit for diagnosing thepresence or absence of a dystonia disorder in a human comprising one ormore reagents for detecting a mutation in a dystonia gene, such as DYT1, or a dystonia protein, such as TOR-A, in a sample obtained from thehuman. The one or more reagents for detecting the torsion dystonia areused for carrying out an enzyme-linked immunosorbent assay or aradioimmunoassay to detect the presence of absence of dystonia protein.In another embodiment the kit comprises one or more reagents fordetecting the torsion dystonia by carrying out a PCR, hybridization orsequence-based assay or any combination thereof.

[0033] It is also envisioned that the methods of the present inventioncan diagnosis a mutation in a dystonia gene, such as DYT1, which encodesa dystonia protein, such as TOR-A, wherein a mutation in the dystoniagene for the human is compared to a mutation in a dystonia gene for aparent of the human who is unaffected by a torsion dystonia, a parent ofthe human who is affected by the torsion dystonia and a sibling of thehuman who is affected by the torsin dystonia.

[0034] The invention also provides methods for therapeutic usesinvolving all or part of the nucleic acid sequence encoding torsinprotein or torsin protein.

[0035] The invention further provides nucleic acid sequences useful asprobes and primers for the identification of mutations or polymorphismswhich mediate clinical neuronal diseases, or which confer increasedvulnerability (e. g., genetic predisposition) respectively, to otherneuronal diseases.

[0036] Another embodiment of the invention provides methods utilizingthe disclosed probes and primers to detect mutations or polymorphisms inother neuronal genes implicated in conferring a particular phenotypewhich gives rise to overt clinical symptoms in a mammal that areconsistent with (e. g., correlate with) the neuroanatomical expressionof the gene. For example, the methods described herein can be used toconfirm the role of TOR-1, TOR-2, ooc-5, TOR-A or TOR-B in neuronaldiseases, including but not limited to dopamine-mediated diseases,movement disorders, neurodegenerative diseases, neurodevelopmentaldiseases and neuropsychiatric disorders.

[0037] An particular embodiment provides a method of identifying a genecomprising a mutation or a polymorphism resulting in a dopamine-mediateddisease, or a neuronal disease. Examples of such diseases arerepresented in Table 1.

[0038] Another embodiment of the invention provides a method ofidentifying a mutation or polymorphism in a neuronal gene which confersincreased susceptibility to a neuronal disease.

[0039] Another object of the present invention is a method of reducing,arresting, alleviating, ameliorating, or preventing protein aggregationin the presence of a torsin protein relative to a level of proteinaggregation in the absence of the torsin protein. The torsin protein maybe mutated. This method may be conducted in the presence of furthercompounds that of reducing, arresting, alleviating, ameliorating, orpreventing protein aggregation

[0040] Another object of the present invention is a method of reducing,arresting, alleviating, ameliorating, or preventing cellular dysfunctionas a result of protein aggregation. This method may be conducted in thepresence of further compounds that of reducing, arresting, alleviating,ameliorating, or preventing cellular dysfunction as a result of proteinaggregation.

[0041] Another object of the present invention is a method of treating,reducing, arresting, alleviating, ameliorating, or preventingprotein-aggregation-associated diseases. Examples ofprotein-aggregation-associated diseases are those represented inTable 1. This method may be conducted in the presence of furthercompounds that of reducing, arresting, alleviating, ameliorating, orpreventing protein-aggregation-associated diseases.

[0042] Another object of the present invention is a method of treating,reducing, arresting, alleviating, ameliorating, or preventing symptomsof protein-aggregation-associated diseases. Examples ofprotein-aggregation-associated diseases are those represented inTable 1. This method may be conducted in the presence of furthercompounds that of reducing, arresting, alleviating, ameliorating, orpreventing symptoms of protein-aggregation-associated diseases.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIG. 1: A polynucleotide sequence alignment of tor-2 vs. DYT1.

[0044]FIG. 2: A polynucleotide sequence alignment of tor-2 vs. DYT2.

[0045]FIG. 3: A polypeptide sequence alignment of TOR-1, TOR-2, OOC-5,TOR-A, and TOR-B.

[0046]FIG. 4a: Expression of 19 polyglutamine repeats (Q19).

[0047]FIG. 4b: Expression of 82 polyglutamine repeats (Q82).

[0048]FIG. 4c: Co-expression of Q82 and tor-2.

[0049]FIG. 4d: Co-expression of Q82 and tor-2/Δ368.

[0050]FIG. 5: Size of Q82 aggregates.

[0051]FIG. 6a: Tail pictures of Q82, Q82+tor-2, and Q82+tor-2/66 368.

[0052]FIG. 6b: Close-up pictures of Q82, Q82+tor-2, and Q82+tor-2/Δ368.

[0053]FIG. 7: Graph of Q19 aggregate accumulation vs. time.

[0054]FIG. 8: Immunolocalization by whole worm antibody staining withtor-2-specific antibody.

[0055]FIG. 9. Western blot of whole protein extracts from C. eleganswith actin control and tor-2 antibody.

[0056]FIG. 10a: Expression of 82 polyglutamine repeats (Q82).

[0057]FIG. 10b: Co-expression of Q82 and TOR-2.

[0058]FIG. 10c: Co-expression of Q82 and OOC-5.

[0059]FIG. 10d: Co-expression of Q82 and TOR-A.

[0060]FIG. 10e: Co-expression of Q82 and OOC-5 and TOR-2.

DETAILED DESCRIPTION OF THE INVENTION

[0061] Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, New York (2001), Current Protocolsin Molecular Biology, Ausebel et al (eds.), John Wiley & Sons, New York(2001) and the various references cited therein.

[0062] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting. The presentinvention provide torsin proteins and polynucleotides that encode theproteins. Torsin proteins are known to occur in humans and thought tooccur C. elegans. Until now, the function of torsin proteins wascompletely unknown. However, the present invention establishes that atleast one function of torsin proteins is the prevention of proteinaggregation. There are two human torsin proteins, TOR1A and TOR1B, andthere are three torsin proteins from C. elegans, TOR-1, TOR-2, andOOC-5.

[0063] Within the context of the present invention “isolated” or“purified” means separated out of its natural environment, which is alsosubstantially free of other contaminating proteins, polynucleotides,and/or other biological materials often found in cell extracts.

[0064] Within the context of the present invention “Polynucleotide” ingeneral relates to polyribonucleotides and polydeoxyribonucleotides, itbeing possible for these to be non-modified RNA or DNA or modified RNAor DNA.

[0065] “Consisting essentially of”, in relation to a nucleic acidsequence, is a term used hereinafter for the purposes of thespecification and claims to refer to substitution of nucleotides asrelated to third base degeneracy. As appreciated by those skilled in theart, because of third base degeneracy, almost every amino acid can berepresented by more than one triplet codon in a coding nucleotidesequence. Further, minor base pair changes may result in variation(conservative substitution) in the amino acid sequence encoded, are notexpected to substantially alter the biological activity of the geneproduct. Thus, a nucleic acid sequencing encoding a protein or peptideas disclosed herein, may be modified slightly in sequence (e.g.,substitution of a nucleotide in a triplet codon), and yet still encodeits respective gene product of the same amino acid sequence. The aminoacid sequence of TOR-2 is shown as SEQ ID NO:2 and the genomic sequenceencoding the TOR-2 protein is shown as SEQ ID NO:1. The amino acidsequence of TOR-1 is shown as SEQ ID NO:4 and the genomic sequenceencoding the TOR-1 protein is shown as SEQ ID NO:3. The amino acidsequence of OOC-5 is shown as SEQ ID NO:6 and the genomic sequenceencoding the OOC-5 protein is shown as SEQ ID NO:5. The amino acidsequence of TOR-A is shown as SEQ ID NO:8 and the genomic sequenceencoding the TOR-A protein is shown as SEQ ID NO:7. The amino acidsequence of TOR-B is shown as SEQ ID NO:10 and the genomic sequenceencoding the TOR-B protein is shown as SEQ ID NO:9.

[0066] One skilled in the art will realize that organisms other thanhumans will also contain torsin genes (for example, eukaryotes; morespecifically, mammals (preferably, gorillas, rhesus monkeys, andchimpanzees), rodents, worms (preferably, C. elegans), insects(preferably, D. melanogaster) birds, fish, yeast, and plants). Theinvention is intended to include, but is not limited to, torsin nucleicacid molecules isolated from the above-described organisms.

[0067] Isolated nucleic acid molecules of the present invention are alsomeant to include those chemically synthesized. For example, a nucleicacid molecule with the nucleotide sequence which codes for theexpression product of a torsin gene can be designed and, if necessary,divided into appropriate smaller fragments. Then an oligomer whichcorresponds to the nucleic acid molecule, or to each of the dividedfragments, can be synthesized. Such synthetic oligonucleotides can beprepared synthetically (Matteucci et al., 1981, J Am. Chem. Soc.103:3185-3191) or by using an automated DNA synthesizer. Anoligonucleotide can be derived synthetically or by cloning. Ifnecessary, the 5′ ends of the oligonucleotides can be phosphorylatedusing T4 polynucleotide kinase. Kinasing the 5′ end of anoligonucleotide provides a way to label a particular oligonucleotide by,for example, attaching a radioisotope (usually .sup.32p) to the 5′ end.Subsequently, the oligonucleotide can be subjected to annealing andligation with T4 ligase or the like.

[0068] To isolate the torsin genes or also other genes, a gene libraryis first set up. The setting up of gene libraries is described ingenerally known textbooks and handbooks. The textbook by Winnacker: Geneund Klone, Eine Einführung in die Gentechnologie [Genes and Clones, AnIntroduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany,1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kobara et al. (Cell 50,495-508 (1987)).

[0069] To prepare a gene library in E. coli, it is also possible to useplasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) orpUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are, inparticular, those E. coli strains which are restriction-andrecombination-defective, such as the strain DH5αmcr, which has beendescribed by Grant et al. (Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649).

[0070] The long DNA fragments cloned with the aid of cosmids or other λvectors can then in turn be subcloned and subsequently sequenced in theusual vectors which are suitable for DNA sequencing, such as isdescribed e.g. by Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

[0071] The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

[0072] The new torsin sequences for the torsin genes which are relatedto SEQ ID NOS. 2, 4, 6, 8, and 10, is a constituent of the presentinvention has been found in this manner. The amino acid sequence of thecorresponding protein has furthermore been derived from the present DNAsequence by the methods described above. The resulting amino acidsequence of the torsin gene products is shown in SEQ ID NOS. 2, 4, 6, 8,and 10.

[0073] Coding DNA sequences, which result from SEQ ID NOS. 1, 3, 5, 7,and 9 by the degeneracy of the genetic code, are also a constituent ofthe invention. In the same way, DNA sequences, which hybridize with SEQID NOS. 1, 3, 5, 7, and 9 or parts of SEQ ID NOS. 1, 3, 5, 7, and 9, area constituent of the invention. Conservative amino acid exchanges, suchas e.g. exchange of glycine for alanine or of aspartic acid for glutamicacid in proteins, are furthermore known among experts as “sensemutations” which do not lead to a fundamental change in the activity ofthe protein, i.e. are of neutral function. It is furthermore known thatchanges on the N and/or C terminus of a protein cannot substantiallyimpair or can even stabilize the function thereof. Information in thiscontext can be found by the expert, inter alia, in Ben-Bassat et al.(Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247(1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and inknown textbooks of genetics and molecular biology. Amino acid sequences,which result in a corresponding manner from SEQ ID NOS. 2, 4, 6, 8, and10, are also a constituent of the invention.

[0074] In the same way, DNA sequences, which hybridize with SEQ ID NOS.1, 3, 5, 7, and 9 or parts of SEQ ID NOS. 1, 3, 5, 7, and 9, are aconstituent of the invention. Finally, DNA sequences, which are preparedby the polymerase chain reaction (PCR) using primers, which result fromSEQ ID NOS. 1, 3, 5, 7, and 9, are a constituent of the invention. Sucholigonucleotides typically have a length of at least 15 nucleotides.

[0075] The skilled artisan will find instructions for identifying DNAsequences by means of hybridization can be found by the expert, interalia, in the handbook “The DIG System Users Guide for FilterHybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993)and in Liebl et al. (International Journal of Systematic Bacteriology41: 255-260 (1991)). The hybridization takes place under stringentconditions, that is to say only hybrids in which the probe and targetsequence, i. e. the polynucleotides treated with the probe, are at least70% identical are formed. It is known that the stringency of thehybridization, including the washing steps, is influenced or determinedby varying the buffer composition, the temperature and the saltconcentration. The hybridization reaction is preferably carried outunder a relatively low stringency compared with the washing steps(Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0076] A 5×SSC buffer at a temperature of approx. 50° C.-68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides, which are less than 70% identicalto the sequence of the probe. Such hybrids are less stable and areremoved by washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2×SSC and optionallysubsequently 0.5×SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50° C.-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1×SSC.Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% identical to the sequence of the probe employed can beisolated by increasing the hybridization temperature stepwise from 50°C. to 68° C. in steps of approx. 1-2° C. Further instructions onhybridization are obtainable on the market in the form of so-called kits(e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany,Catalogue No. 1603558).

[0077] A skilled artisan will find instructions for amplification of DNAsequences with the aid of the polymerase chain reaction (PCR) can befound by the expert, inter alia, in the handbook by Gait:Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK,1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag,Heidelberg, Germany, 1994).

[0078] A “mutation” is any detectable change in the genetic materialwhich can be transmitted to daughter cells and possibly even tosucceeding generations giving rise to mutant cells or mutantindividuals. If the descendants of a mutant cell give rise only tosomatic cells in multicellular organisms, a mutant spot or area of cellsarises. Mutations in the germ line of sexually reproducing organisms canbe transmitted by the gametes to the next generation resulting in anindividual with the new mutant condition in both its somatic and germcells. A mutation can be any (or a combination of) detectable, unnaturalchange affecting the chemical or physical constitution, mutability,replication, phenotypic function, or recombination of one or moredeoxyribonucleotides; nucleotides can be added, deleted, substitutedfor, inverted, or transposed to new positions with and withoutinversion. Mutations can occur spontaneously and can be inducedexperimentally by application of mutagens. A mutant variation of anucleic acid molecule results from a mutation. A mutant polypeptide canresult form a mutant nucleic acid molecule and also refers to apolypeptide which is modified at one, or more, amino acid residues fromthe wildtype (naturally occurring) polypeptide. The term “mutation”, asused herein, can also refer to any modification in a nucleic acidsequence encoding a dystonia protein. For example, the mutation can be apoint mutation or the addition, deletion, insertion and/or substitutionof one or more nucleotides or any combination thereof. The mutation canbe a missense or frameshift mutation. Modifications can be, for example,conserved or non-conserved, natural or unnatural.

[0079] “Consisting essentially of”, in relation to amino acid sequenceof a protein or peptide, is a term used hereinafter for the purposes ofthe specification and claims to refer to a conservative substitution ormodification of one or more amino acids in that sequence such that thetertiary configuration of the protein or peptide is substantiallyunchanged.

[0080] “Conservative substitutions” is defined by aforementionedfunction, and includes substitutions of amino acids having substantiallythe same charge, size, hydrophilicity, and/or aromaticity as the aminoacid replaced. Such substitutions, known to those of ordinary skill inthe art, include glycine-alanine-valine; isoleucine-leucine;tryptophan-tyrosine; aspartic acid-glutamic acid; arginine-lysine;asparagine-glutamine; and serine-threonine. “modification”, in relationto amino acid sequence of a protein or peptide, is defined functionallyas a deletion of one or more amino acids which does not impart a changein the conformation, and hence the biological activity, of the proteinor peptide sequence.

[0081] The term “expression vector” refers to an polynucleotide thatencodes the torsin proteins or fragments thereof of the invention andprovides the sequences necessary for its expression in the selected hostcell. The recombinant host cells of the present invention may bemaintained in vitro, e.g., for recombinant protein, polypeptide orpeptide production. Equally, the recombinant host cells could be hostcells in vivo, such as results from immunization of an animal or humanwith a nucleic acid segment of the invention. Accordingly, therecombinant host cells may be prokaryotic or eukaryotic host cells, suchas E. coli, Saccharomyces cerevisiae or other yeast, mammalian or humanhost cells. Expression vectors will generally include a transcriptionalpromoter and terminator, or will provide for incorporation adjacent toan endogenous promoter. Expression vectors will usually be plasmids,further comprising an origin of replication and one or more selectablemarkers. However, expression vectors may alternatively be viralrecombinants designed to infect the host, or integrating vectorsdesigned to integrate at a preferred site within the host's genome.Examples of other expression vectors are disclosed in Molecular Cloning:A Laboratory Manual, Third Edition, Sambrook, Fritsch, and Maniatis,Cold Spring Harbor Laboratory Press, 2001. In a preferred embodimentthese polynucleotides that hybridize under stringent conditions alsoencode a protein or peptide which has torsin activity.

[0082] “Torsin activity” within the context of the present inventionincludes reducing, alleviating, arresting, ameliorating, and inhibitingprotein aggregation.

[0083] “Torsin gene” within the context of the present inventionincludes any polynucleotide encoding a polypeptide having torsinactivity.

[0084] “Torsin protein” within the context of the present inventionincludes any polypeptide having torsin activity.

[0085] The common amino acids are generally known in the art. Additionalamino acids that may be included in the peptide of the present inventioninclude: L-norleucine; aminobutyric acid; L-homophenylalanine;L-norvaline; D-alanine; D-cysteine; D-aspartic acid; D-glutamic acid;D-phenylalanine; D-histidine; D-isoleucine; D-lysine; D-leucine;D-methionine; D-asparagine; D-proline; D-glutamine; D-arginine;D-serine; D-threonine; D-valine; D-tryptophan; D-tyrosine; D-omithine;aminoisobutyric acid; L-ethylglycine; L-t-butylglycine; penicillamine;I-naphthylalanine; cyclohexylalanine; cyclopentylalanine;aminocyclopropane carboxylate; aminonorbomylcarboxylate;L-α-methylalanine; L-α-methylcysteine; L-α-methylaspartic acid;L-α-methylglutamic acid; L-α-methylphenylalanine; L α-methylhistidine;L-α-methylisoleucine; L-α-methyllysine; L-α-methylleucine;L-α-methylmethionine; L-α-methylasparagine; L-α-methylproline;L-α-methylglutamine; L-α-methylarginine; L-α-methylserine;L-α-methylthreonine; L-α-methylvaline; L-α-methyltryptophan;L-α-methyltyrosine; L-α-methylomithine; L-α-methylnorleucine;amino-α-methylbutyric acid;. L-α-methylnorvaiine;L-α-methylhomophenylalanine; L-α-methylethylglycine;methyl-α-aminobutyric acid;. methylaminoisobutyric acid;L-α-methyl-t-butylglycine; methylpenicillamine;methyl-α-naphthylalanine; methylcyclohexylalanine;methylcyclopentylalanine; D-α-methylalanine; D-α-methylomithine;D-α-methylcysteine; D-α-methylaspartic acid; D-α-methylglutamic acid;D-α-methylphenylalanine; D-α-methylhistidine; D-α-methylisoleucine;D-α-methyllysine; D-α-methylleucine; D-α-methylmethionine; D--methylasparagine; D-α-methylproline; D-α-methylglutamine;D-α-methylarginine; D-α-methylserine; D-α-methylthreonine;D-α-methylvaline; D-α-methyltryptophan; D-α-methyltyrosine;L-N-methylalanine; L-N-methylcysteine; L-N-methylaspartic acid;L-N-methylglutamic acid; L-N-methylphenylalanine; L-N-methylhistidine;L-N-methylisoleucine; L-N-methyllysine; L-N-methylleucine;L-N-methylmethionine; L-N-methylasparagine; N-methylcyclohexylalanine;L-N-methylglutamine; L-N-methylarginine; L-N-methylserine;L-N-methylthreonine; L-N-methylvaline; L-N-methyltryptophan;L-N-methyltyrosine; L-N-methylomithine; L-N-methylnorleucine;N-aminoα-methylbutyric acid; L-N-methylnorvaiine;L-N-methylhomophenylalanine; L-N-methylethylglycine;N-methyl-yaminobutyric acid; N-methylcyclopentylalanine;L-N-methyl-t-butylglycine; N-methylpenicillamine;N-methyl-a-naphthylalanine; N-methylaminoisobutyric acid;N-(2-aminoethyl)glycine; D-N-methylalanine; D-N-methylomithine;D-N-methylcysteine; D-N-methylaspartic acid; D-N-methylglutamic acid;D-N-methylphenylalanine; D-N-methylhistidine; D-N-methylisoleucine;D-N-methyllysine; D-N-methylleucine; D-N-methylmethionine;D-N-methylasparagine; D-N-methylproline; D-N-methylglutamine;D-N-methylarginine; D-N-methylserine; D-N-methylthreonine;D-N-methylvaline; D-N-methyltryptophan; D-N-methyltyrosine;N-methylglycine; N-(carboxymethyl)glycine; N-(2-carboxyethyl)glycine;N-benzylglycine; N-(imidazolylethyl)glycine; N-(1-methylpropyl)glycine;N-(4-aminobutyl)glycine; N-(2-methylpropyl)glycine;N-(2-methylthioethyl)glycine; N-(hydroxyethyl)glycine;N-(carbamylmethyl)glycine; N-(2-carbamylethyl)glycine;N-(1-methylethyl)glycine; N-(3-guanidinopropyl)glycine;N-(3-indolylethyl)glycine; N-(p-hydroxyphenethyl)glycine;N-(1-hydroxyethyl)glycine; N-(thiomethyl)glycine;N-(3-aminopropyl)glycine; N-cyclopropylglycine; N-cyclobutyglycine;N-cyclohexylglycine; N-cycloheptylglycine; N-cyclooctylglycine;N-cyclodecylglycine; N-cycloundecylglycine; N-cyclododecylglycine;N-(2,2-diphenylethyl)glycine; N-(3,3-diphenylpropyl)glycine;N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine;N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine; and1-carboxy-1-(2,2-diphenylethylamino)cyclopropane.

[0086] Because its amino acid sequence has been disclosed by the presentinvention, the TOR-1 and TOR-2 proteins or fragments thereof of thepresent invention can be produced by a known chemical synthesis method(for example, a liquid phase synthesis method, a solid phase synthesismethod, and others.; Izumiya, N., Kato, T., Aoyagi, H., Waki, M., “Basisand Experiments of Peptide Synthesis”, 1985, Maruzen Co., Ltd.) based onthat sequence. Typically, peptide synthesis is carried out for shorterpeptide fragments of about 100 amino acids or less.

[0087] The TOR-1 and TOR-2 proteins or fragments thereof of the presentinvention may contain one or more protected amino acid residues. Theprotected amino acid is an amino acid whose functional group or groupsis/are protected with a protecting group or groups by a known method andvarious protected amino acids are commercially available.

[0088] The TOR-1 and TOR-2 proteins or fragments thereof of the presentinvention may be provided in a glycosylated as well as an unglycosylatedform. Preparation of glycosylated TOR-1 and TOR-2 proteins or fragmentsthereof is known in the art and typically involves expression of therecombinant DNA encoding the peptide in a eukaryotic cell. Likewise, itis generally known in the art to express the recombinant DNA encodingthe peptide in a prokaryotic (e.g., bacterial) cell to obtain a peptide,which is not glycosylated. These and other methods of alteringcarbohydrate moieties on glycoproteins is found, inter alia, inEssentials of Glycobiology (1999), Edited By Ajit Varki, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., the contents of whichare incorporated herein by reference.

[0089] Alternatively, the TOR-1 and TOR-2 proteins or fragments thereofof the present invention can be produced by producing a polynucleotide(DNA or RNA) which corresponds to the amino acid sequence of the TOR-1and TOR-2 proteins or fragments thereof of the present invention andproducing the TOR-1 and TOR-2 proteins or fragments thereof by a geneticengineering technique using the polynucleotide. Polynucleotide codingsequences for amino acid residues are known in the art and are disclosedfor example in Molecular Cloning: A Laboratory Manual, Third Edition,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press,2001.

[0090] In another embodiment, the present invention relates to apurified polypeptide preferably, substantially pure) having an aminoacid sequence corresponding to a torsin protein, or a functionalderivative thereof. In a preferred embodiment, the polypeptide has theamino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, and 10 ormutant or species variation thereof, or at least 70% identity, furtherat least 80% identity or and even further at least 90% identity thereof(preferably, at least 90%, 95%, 96%, 97%, 98%, or 99% identity or atleast 95%, 96%, 97%, 98%, or 99% similarity thereof), or at least 6contiguous amino acids thereof (preferably, at least 10, 15, 20, 25, or50 contiguous amino acids thereof).

[0091] In a preferred embodiment, the invention relates to torsinepitopes. The epitope of these polypeptides is an immunogenic orantigenic epitope. An immunogenic epitope is that part of the proteinwhich elicits an antibody response when the whole protein is theimmunogen. An antigenic epitope is a fragment of the protein which canelicit an antibody response. Methods of selecting antigenic epitopefragments are well known in the art (Sutcliffe et al., 1983, Science.219:660-666). Antigenic epitope-bearing peptides and polypeptides of theinvention are useful to raise an immune response that specificallyrecognizes the polypeptides. Antigenic epitope-bearing peptides andpolypeptides of the invention comprise at least 7 amino acids(preferably, 9, 10, 12, 15 or 20 amino acids) of the proteins of theAmino acid sequence variants of torsin can be prepared by mutations inthe DNA. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequenceshown in SEQ ID NOS: 2, 4, 6, 8, and 10. Any combination of deletion,insertion, and substitution can also be made to arrive at the finalconstruct, provided that the final construct possesses the desiredactivity. While the site for introducing an amino acid sequencevariation is predetermined, the mutation itself need not bepredetermined. For example, to optimize the performance of a particularpolypeptide with respect to a desired activity, random mutagenesis canbe conducted at a target codon or region of the polypeptide, and theexpressed variants can be screened for the optimal desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, e.g., site-specificmutagenesis.

[0092] Preparation of a torsin variant in accordance herewith ispreferably achieved by site-specific mutagenesis of DNA that encodes anearlier prepared variant or a non-variant version of the protein.Site-specific mutagenesis allows the production of torsin variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation. In general, the technique ofsite-specific mutagenesis is well known in the art (Adelman et al.,1983, DNA 2:183; Ausubel, et al., In: Current Protocols in MolecularBiology, John Wiley & Sons, (1998)).

[0093] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues.

[0094] Amino acid sequence insertions include amino and/or carboxylterminal fusions from one residue to polypeptides of essentiallyunrestricted length, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions, (i.e.,insertions within the complete torsin sequence) can range generally fromabout 1 to 10 residues, more preferably 1 to 5.

[0095] The third group of variants are those in which at least one aminoacid residue in the torsin molecule, and preferably, only one, has beenremoved and a different residue inserted in its place.

[0096] Substantial changes in functional or immunological identity aremade by selecting substitutions that are less conservative, i.e.,selecting residues that differ more significantly in their effect onmaintaining a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, b)the charge or hydrophobicity of the molecule at the target site, or c)the bulk of the side chain. The substitutions that in general areexpected are those in which a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; c) a cysteine residueis substituted for any other residue; d) a residue having anelectropositive side chain, e.g., lysyl, arginyl, or histidyl, issubstituted for a residue having an electronegative charge, e.g.,glutamyl or aspartyl; or e) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for one not having such a side chain,e.g., glycine.

[0097] Some deletions, insertions and substitutions are not expected toproduce radical changes in the characteristics of torsin. However, whileit is difficult to predict the exact effect of the deletion, insertionor substitution in advance, one skilled in the art will appreciate thatthe effect can be evaluated by biochemical and in vivo screening assays.For example, a variant typically is made by site-specific mutagenesis ofthe native torsin-encoding nucleic acid, expression of the variantnucleic acid in cell culture, and, optionally, purification from thecell culture, for example, by immunoaffinity adsorption on a column (toabsorb the variant by binding it to at least one immune epitope). Theactivity of the cell culture lysate or purified torsin variant is thenscreened by a suitable screening assay for the desired characteristic.For example, a change in the immunological character of the torsinmolecule, such as affinity for a given antibody, can be measured by acompetitive type immunoassay. Changes in immunomodulation activity canbe measured by the appropriate assay. Modifications of such proteinproperties as redox or thermal stability, enzymatic activity,hydrophobicity, susceptibility to proteolytic degradation or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to those of ordinary skill in the art.

[0098] A variety of methodologies known in the art can be utilized toobtain the polypeptide of the present invention. In one embodiment, thepolypeptide is purified from tissues or cells which naturally producethe peptide. Alternatively, the above described isolated nucleic acidfragments can be used to express the torsin protein in any organism. Thesamples of the present invention include cells, protein extracts ormembrane extracts of cells, or biological fluids. The sample will varybased on the assay format, the detection method and the nature of thetissues, cells or extracts used as the sample

[0099] Any organism can be used as a source for the polypeptide of theinvention, as long as the source organism naturally contains such apeptide. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence of the polypeptide isderived, regardless of the organism the polypeptide is expressed in andultimately isolated from.

[0100] One skilled in the art can readily follow known methods forisolating proteins in order to obtain the polypeptide free of naturalcontaminants. These include, but are not limited to:immunochromotography, size-exclusion chromatography, ion-exchangechromatography, hydrophobic interaction chromatography, andnon-chromatographic separation methods.

[0101] In a preferred embodiment, the purification procedures compriseion-exchange chromatography and size exclusion chromatography. Any of alarge number of ion-exchange resins known in the art can be employed,including, for example, monoQ, Sepharose-Q, macro-prepQ, AG1-X2, or HQ.Examples of suitable size exclusion resins include, but are not limitedto, Superdex 200, Superose 12, and Sephycryl 200. Elution can beachieved with aqueous solutions of potassium chloride or sodium chlorideat concentrations ranging from 0.01 M to 2. OM over a wide range of pH.

[0102] In another embodiment, the present invention relates to a nucleicacid probe for the specific detection of the presence of torsin nucleicacid in a sample comprising the above-described nucleic acid moleculesor at least a fragment thereof which hybridizes under stringenthybridization and wash conditions to torsin nucleic acid.

[0103] In one preferred embodiment, the present invention relates to anisolated nucleic acid probe consisting of 10 to 1000 nucleotides(preferably, 10 to 500, 10 to 100, 10 to 50, 10 to 35, 20 to 1000, 20 to500, 20 to 100, 20 to 50, or 20 to 35) which hybridizes preferentiallyto torsin RNA or DNA, wherein said nucleic acid probe is or iscomplementary to a nucleotide sequence consisting of at least 10consecutive nucleotides (preferably, 15, 18, 20, 25, or 30) from thenucleic acid molecule comprising a polynucleotide sequence at least 90%identical to one or more of the following: a nucleotide sequenceencoding a torsin polypeptide (for example, those described by SEQ IDNOS: 2, 4, 6, 8, and 10); a nucleotide sequence complementary to any ofthe above nucleotide sequences; and any nucleotide sequence aspreviously described above.

[0104] The nucleic acid probe can be used to probe an appropriatechromosomal or cDNA library by usual hybridization methods to obtainanother nucleic acid molecule of the present invention. A chromosomalDNA or cDNA library can be prepared from appropriate cells according torecognized methods in the art (Sambrook, J., Fritsch, E. F., andManiatis, T., 1989, In: Molecular Cloning. A Laboratory Manual., ColdSpring Harbor Laboratory Press, Cold Spring Harbor).

[0105] In the alternative, chemical synthesis is carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the torsin amino acid sequence.Thus, the synthesized nucleic acid probes can be used as primers in apolymerase chain reaction (PCR) carried out in accordance withrecognized PCR techniques (PCR Protocols, A Guide to Methods andApplications, edited by Michael et al., Academic Press, 1990), utilizingthe appropriate chromosomal, cDNA or cell line library to obtain thefragment of the present invention.

[0106] The hybridization probes of the present invention can be labeledfor detection by standard labeling techniques such as with aradiolabeling, fluorescent labeling, biotin-avidin labeling,chemiluminescence, and the like. After hybridization, the probes can bevisualized using known methods.

[0107] The nucleic acid probes of the present invention include RNA, aswell as DNA probes, such probes being generated using techniques knownin the art.

[0108] In one embodiment of the above described method, a nucleic acidprobe is immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art.

[0109] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the described methodswill vary based on the assay format, the detection method and the natureof the tissues, cells or extracts used in the assay. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

[0110] In another embodiment, the present invention relates to a methodof detecting the presence of torsin nucleic acid in a sample bycontacting the sample with the above-described nucleic acid probe, underspecific hybridization conditions such that hybridization occurs, anddetecting the presence of the probe bound to the nucleic acid molecule.One skilled in the art would select the nucleic acid probe according totechniques known in the art as described above. Samples to be testedinclude but should not be limited to RNA or DNA samples from humantissue.

[0111] In another embodiment, the present invention relates to a kit fordetecting, in a sample, the presence of a torsin nucleic acid. The kitcomprises at least one container having disposed therein theabove-described nucleic acid probe. In a preferred embodiment, the kitfurther comprises other containers comprising wash reagents and/orreagents capable of detecting the presence of the hybridized nucleicacid probe. Examples of detection reagents include, but are not limitedto radiolabeled probes, enzymatic probes (horseradish peroxidase,alkaline phosphatase), and affinity labeled probes (biotin, avidin, orstreptavidin).

[0112] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris buffers, and the like), and containers whichcontain the reagents used to detect the hybridized probe, boundantibody, amplified product, or the like.

[0113] One skilled in the art will readily recognize that the nucleicacid probes described in the present invention can readily beincorporated into one of the established kit formats which are wellknown in the art.

[0114] In another embodiment, the present invention relates to arecombinant DNA molecule comprising, 5′ to 3′, a promoter effective toinitiate transcription in a host cell and the above-described nucleicacid molecules. In another embodiment, the present invention relates toa recombinant DNA molecule comprising a vector and an above-describednucleic acid molecule.

[0115] In another embodiment, the present invention relates to a nucleicacid molecule comprising a transcriptional control region functional ina cell, a sequence complementary to an RNA sequence encoding an aminoacid sequence corresponding to the above-described polypeptide, and atranscriptional termination region functional in the cell.

[0116] Preferably, the above-described molecules are isolated and/orpurified DNA molecules.

[0117] In another embodiment, the present invention relates to a cell ornon-human organism that contains an above-described nucleic acidmolecule.

[0118] In another embodiment, the peptide is purified from cells whichhave been altered to express the peptide.

[0119] As used herein, a cell is said to be “altered to express adesired peptide” when the cell, through genetic manipulation, is made toproduce a protein which it normally does not produce or which the cellnormally produces at low levels. One skilled in the art can readilyadapt procedures for introducing and expressing either genomic, cDNA, orsynthetic sequences into either eukaryotic or prokaryotic cells.

[0120] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression. The precise natureof the regulatory regions needed for gene expression can vary fromorganism to organism, but shall in general include a promoter regionwhich, in prokaryotes for example, contains both the promoter, whichdirects the initiation of RNA transcription, as well as the DNAsequences that, when transcribed into RNA, will signal translationalinitiation. Such regions will normally include those 5′ non-codingsequences involved with initiation of transcription and translation,such as the TATA box, capping sequence, CAAT sequence, and the like.

[0121] If desired, the non-coding region 3′ to the torsin codingsequence can be obtained by the above-described methods. This region canbe retained for its transcriptional termination regulatory sequences,such as termination and polyadenylation signals. Thus, by retaining the3′ region naturally contiguous to the DNA sequence encoding a torsingene, the transcriptional termination signals are provided. Where thetranscriptional termination signals are not functional in the expressionhost cell, then a functional 3′ region derived from host sequences canbe substituted.

[0122] Two DNA sequences (such as a promoter region sequence and antorsin coding sequence) are said to be operably linked if the nature ofthe linkage between the two DNA sequences does not (1) result in theintroduction of a frameshift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription of a torsin codingsequence, or (3) interfere with the ability of the torsin codingsequence to be transcribed by the promoter. Thus, a promoter regionwould be operably linked to a DNA sequence if the promoter were capableof effecting transcription of that DNA sequence.

[0123] The present invention encompasses the expression of the torsincoding sequence (or a functional derivative thereof) in eitherprokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, themost efficient and convenient for the production of recombinantproteins. Prokaryotes most frequently are represented by various strainsof E. coli, however other microbial strains can also be used, includingother bacterial strains such as those belonging to bacterial familiessuch as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, andthe like. In prokaryotic systems, plasmid vectors that containreplication sites and control sequences derived from a speciescompatible with the host can be used. Examples of suitable plasmidvectors include pBR322, pUC18, pUC19, pUC118, pUC119 and the like;suitable phage or bacteriophage vectors include .lambda.gt10,.lambda.gt11 and the like. For eukaryotic expression systems, suitableviral vectors include pMAM-neo, pKRC and the like. Preferably, theselected vector of the present invention has the capacity to replicatein the selected host cell.

[0124] To express torsin in a prokaryotic cell, it is necessary tooperably link the torsin coding sequence to a functional prokaryoticpromoter. Such promoters can be either constitutive or, more preferably,regulatable (i.e., inducible or derepressible). Examples of constitutivepromoters include the in promoter of bacteriophage lambda., the blapromoter of the .beta.-lactamase gene, and the CAT promoter of thechloramphenicol acetyl transferase gene, and the like. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage lambda. (P.sub.L and P.sub.R), the trp, recA,lacZ lacI, and gal promoters of E. coli, the .alpha.-amylase (Ulmanen etal., 1985, J. Bacteriol. 162:176-182) and the .zeta.-28-specificpromoters of B. subtilis (Gilman et al., 1984, Gene sequence 32:11-20),the promoters of the bacteriophages of B. subtilis (Gryczan, In: TheMolecular Biology of the Bacilli, Academic Press, Inc., N.Y. (1982)),and Streptomyces promoters (Ward, et al., 1986, Mol. Gen. Genet.203:468-478).

[0125] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the genesequence-encoding sequence (Gold et al., 1981, Ann. Rev. Microbiol.35:365-404).

[0126] The selection of control sequences, expression vectors,transformation methods, and the like, is dependent on the type of hostcell used to express the gene. The terms “transformants” or “transformedcells” include the primary subject cell and cultures derived therefrom,without regard to the number of transfers. It is also understood thatall progeny cannot be precisely identical in DNA content, due todeliberate or inadvertent mutations. However, as defined, mutant progenyhave the same functionality as that of the originally transformed cell.

[0127] Host cells which can be used in the expression systems of thepresent invention are not strictly limited, provided that they aresuitable for use in the expression of the torsin peptide of interest.Suitable hosts include eukaryotic cells. Preferred eukaryotic hostsinclude, for example, yeast, fungi, insect cells, mammalian cells eitherin vivo, or in tissue culture. Preferred mammalian cells include HeLacells, cells of fibroblast origin such as VERO or CHO-K1, or cells oflymphoid origin and their derivatives.

[0128] In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells, such as the cauliflower mosaicvirus 35S and 19S, nopaline synthase promoter and polyadenylation signalsequences are available.

[0129] Another preferred host is an insect cell, for example Drosophilamelanogaster larvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, 1988, Science.240:1453-1459). Alternatively, baculovirus vectors can be engineered toexpress large amounts of torsin protein in insect cells (Jasny, 1987,Science. 238:1653; Miller et al., In: Genetic Engineering (1986),Setlow, J. K., et al., Eds., Plenum, Vol. 8, pp. 277-297).

[0130] Another example of a host cell is that of within C. elegans.Examples of controlling expression within C. elegans include RNAinterference (RNAi). Fire et al. have described that feeding C. eleganspolynucleotides similar to that of the gene to be expressed can resultin the attenuation of that gene's expression. The literature is full ofreferences describing the many methods to control the expression of agene through RNAi (See for example, U.S. Pat. Nos. 6,355,415, 6,326,193,6,278,039, 6,274,630, 6,266,560, 6,255,071, 6,190,867, 6,025,192,5,837,503, 5,726,299, 5,714,323, 5,693,781, 5,616,459, 5,565,333,5,418,149, 5,198,346, 5,096,815, and 5,015,573).

[0131] Different host cells have characteristic and specific mechanismsfor the translational and post-translational processing and modification(e.g., glycosylation and cleavage) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification of theforeign protein expressed.

[0132] Any of a series of yeast gene expression systems can be utilizedwhich incorporate promoter and termination elements from the activelyexpressed gene sequences coding for glycolytic enzymes. These enzymesare produced in large quantities when yeast are grown in mediums rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals.

[0133] Yeast provides substantial advantages over prokaryotes in that itcan perform post-translational peptide modifications. A number ofrecombinant DNA strategies exist which utilize strong promoter sequencesand high copy number of plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences oncloned mammalian gene products and secretes peptides bearing leadersequences (i.e., pre-peptides).

[0134] For a mammalian host, several possible vector systems areavailable for the expression of torsin. A wide variety oftranscriptional and translational regulatory sequences can be employed,depending upon the nature of the host. The transcriptional andtranslational regulatory signals can be derived from viral sources, suchas adenovirus, bovine papilloma virus, simian virus, or the like, wherethe regulatory signals are associated with a particular gene which has ahigh level of expression. Alternatively, promoters from mammalianexpression products, such as actin, collagen, myosin, and the like, canbe employed. Transcriptional initiation regulatory signals can beselected which allow for repression or activation, so that expression ofthe gene sequences can be modulated. Of interest are regulatory signalswhich are temperature-sensitive so that by varying the temperature,expression can be repressed or initiated, or are subject to chemical(such as metabolite) regulation.

[0135] Expression of torsin in eukaryotic hosts requires the use ofeukaryotic regulatory regions. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis.Preferred eukaryotic promoters include, for example, the promoter of themouse metallothionein I gene sequence (Hamer, et al., 1982, J. Mol.Appi. Gen. 1:273-288); the TK promoter of herpes virus (McKnight, 1982,Cell. 31:355-365); the SV40 early promoter (Benoist, et al., 1981,Nature. 290:304-310); the yeast gal4 gene promoter (Johnston, et al.,1982, Proc. Nat. Acad Sci. USA 79:6971-6975; Silver, et al., 1984, Proc.Natl. Acad. Sci. USA 81:595 1 5955) and the CMV immediate-early genepromoter (Thomsen, et al., 1984, Proc. Natl. Acad. Sci. USA 81:659-663).

[0136] As is widely known, translation of eukaryotic mRNA is initiatedat a codon which encodes methionine. For this reason, it is preferableto ensure that the linkage between a eukaryotic promoter and a torsincoding sequence does not contain any intervening codons which arecapable of encoding a methionine (i.e., AUG). The presence of suchcodons results either in a formation of a fusion protein (if the AUGcodon is in the same reading frame as the torsin coding sequence) or aframe-shift mutation (if the AUG codon is not in the same reading frameas the torsin coding sequence).

[0137] A torsin nucleic acid molecule and an operably linked promotercan be introduced into a recipient prokaryotic or eukaryotic cell eitheras a non-replicating DNA (or RNA) molecule, which can either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the gene can occur through the transient expression of the introducedsequence. Alternatively, permanent expression can occur through theintegration of the introduced DNA sequence into the host chromosome

[0138] In one embodiment, a vector is employed which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected on the basis of one or more markers whichallow for selection of host cells which contain the expression vector.Such markers can provide, for example, for autotrophy to an auxotrophichost or for biocide resistance, e.g., to antibiotics or to heavy metalpoisoning, such as by copper, or the like. The selectable marker genesequence can either be contained on the vector of the DNA gene to beexpressed, or introduced into the same cell by co-transfection.Additional elements might also be necessary for optimal synthesis ofmRNA. These elements can include splice signals, as well astranscription promoters, enhancer signal sequences, and terminationsignals. cDNA expression vectors incorporating such elements have beendescribed (Okayama, 1983, Molec. Cell Biol. 3:280).

[0139] In a preferred embodiment, the introduced nucleic acid moleculewill be incorporated into a plasmid or viral vector capable ofautonomous replication in the recipient host. Any of a wide variety ofvectors can be employed for this purpose. Factors of importance inselecting a particular plasmid or viral vector include: the ease withwhich recipient cells that contain the vector can be recognized andselected from those recipient cells that do not contain the vector; thedesired number of copies of the vector present in the host cell; and theability to “shuttle” the vector between host cells of different species,i.e., between mammalian cells and bacteria. Preferred prokaryoticvectors include plasmids such as those capable of replication in E. coli(for example, pBR322, Co1E1, pSC101, pACYC 184, and .pi.VX). Suchplasmids are commonly known to those of skill in the art (Sambrook, J.,Fritsch, E. F., and Maniatis, T., 1989, In: Molecular Cloning. ALaboratory Manual., Cold Spring Harbor Laboratory Press, Cold SpringHarbor). B. subtilis derived plasmids include pC194, pC221, pT127, andthe like (Gryczan, In: The Molecular Biology of the Bacilli, AcademicPress, NY (1982), pp. 307-329). Suitable Streptomyces plasmids includepIJ101 (Kendall, et al., 1987, J. Bacteriol. 169:4177-4183), andstreptomyces bacteriophages such as .phi.C31 (Chater, et al., In: SixthInternational Symposium on Actinomycetales Biology, Akademiai Kaido,Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids have alsobeen described (John, et al., 1986, Rev. Infect. Dis. 8:693-704; Izaki,1978, Jpn. J Bacteriol. 33:729-742).

[0140] Preferred eukaryotic plasmids include, for example, BPV,vaccinia, SV40, 2 .mu. circle, and the like, or their derivatives. Suchplasmids are well known in the art (Botstein, et al., 1982, Miami Wntr.Symp. 19:265-274; Broach, In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, 1982,Cell. 28:203-204; Bollon, et al, 1980, J. Clin. Hematol. Oncol.10:39-48; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).

[0141] Once the vector or nucleic acid molecule containing the constructhas been prepared for expression, the DNA construct can be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, lipofection, conjugation, protoplastfusion, electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium that allows for selection of vector containing cells. Expressionof the cloned gene results in the production of torsin. This can takeplace in the transformed cells as such, or following the induction ofthese cells to differentiate (for example, by administration ofbromodeoxyuracil to neuroblastoma cells or the like).

[0142] In another embodiment, the present invention relates to anantibody having binding affinity specifically to a torsin polypeptide asdescribed above or specifically to a torsin polypeptide binding fragmentthereof. An antibody binds specifically to a torsin polypeptide orbinding fragment thereof if it does not bind to non-torsin polypeptides.Those which bind selectively to torsin would be chosen for use inmethods which could include, but should not be limited to, the analysisof altered torsin expression in tissue containing torsin.

[0143] The torsin proteins of the present invention can be used in avariety of procedures and methods, such as for the generation ofantibodies, for use in identifying pharmaceutical compositions, and forstudying DNA/protein interaction.

[0144] The torsin peptide of the present invention can be used toproduce antibodies or hybridomas. One skilled in the art will recognizethat if an antibody is desired, such a peptide would be generated asdescribed herein and used as an immunogen.

[0145] The antibodies of the present invention include monoclonal andpolyclonal antibodies, as well as fragments of these antibodies. Theinvention further includes single chain antibodies. Antibody fragmentswhich contain the idiotype of the molecule can be generated by knowntechniques. For example, such fragments include but are not limited to:the F(ab′).sub.2 fragment; the Fab′ fragments, Fab fragments, and Fvfragments.

[0146] Of special interest to the present invention are antibodies totorsin which are produced in humans, or are “humanized” (i.e.,non-immunogenic in a human) by recombinant or other technology.Humanized antibodies can be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e., chimeric antibodies (Robinson, R. R., etal., International Patent Publication PCT/US86/02269; Akira, K., et al.,European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L., et al., European PatentApplication 173,494; Neuberger, M. S., et al., PCT Application WO86/01533; Cabilly, S., et al., European Patent Application 125,023;Better, M., et al, 1988, Science. 240:1041-1043; Liu, A. Y., et al.,1987, Proc. Natl. Acad. Sci. USA. 84:3439-3443; Liu, A. Y., et al.,1987, J. Immunol. 139:3521-3526; Sun, L. K., et al., 1987, Proc. Natl.Acad. Sci. USA 84:214-218; Nishimura, Y., et al., 1987, Canc. Res.47:999-1005; Wood, C. R., et al., 1985, Nature. 314:446-449); Shaw, etal., 1988, J. Natl. Cancer Inst. 80:1553-1559) and “humanized” chimericantibodies (Morrison, S. L., 1985, Science. 229:1202-1207; Oi, V. T., etal., 1986, BioTechniques 4:214)). Suitable “humanized” antibodies can bealternatively produced by CDR or CEA substitution (Jones, P. T., et al.,1986, Nature. 321:552-525; Verhoeyan, et al., 1988, Science. 239:1534;Beidler, C. B., et al., 1988, J. Immunol. 141:4053-4060).

[0147] In another embodiment, the present invention relates to ahybridoma which produces the above-described monoclonal antibody. Ahybridoma is an immortalized cell line which is capable of secreting aspecific monoclonal antibody.

[0148] In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands(1984); St. Groth, et al., 1980, J. Immunol. Methods. 35:1-21).

[0149] The inventive methods utilize antibodies reactive with torsinproteins or portions thereof. In a preferred embodiment, the antibodiesspecifically bind with torsin proteins or a portion or fragment thereof.The antibodies can be polyclonal or monoclonal, and the term antibody isintended to encompass polyclonal and monoclonal antibodies, andfunctional fragments thereof. The terms polyclonal and monoclonal referto the degree of homogeneity of an antibody preparation, and are notintended to be limited to particular methods of production.

[0150] Any animal (mouse, rabbit, and the like) which is known toproduce antibodies can be immunized with the selected polypeptide.Methods for immunization are well known in the art. Such methods includesubcutaneous or intraperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of polypeptide usedfor immunization will vary based on the animal which is immunized, theantigenicity of the polypeptide and the site of injection.

[0151] The polypeptide can be modified or administered in an adjuvant inorder to increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or .beta.-galactosidase) or through the inclusion of anadjuvant during immunization.

[0152] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, and allowed to becomemonoclonal antibody producing hybridoma cells.

[0153] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas by anELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al.,1988, Exp. Cell Res. 175:109-124).

[0154] Hybridomas secreting the desired antibodies are cloned and theclass and subclass is determined using procedures known in the art(Campbell, In: Monoclonal Antibody Technology. Laboratory Techniques inBiochemistry and Molecular Biology, supra (1984)).

[0155] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0156] In another embodiment of the present invention, theabove-described antibodies are detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art (Stemberger, et al., 1970, J. Histochem. Cytochem. 18:315;Bayer, et al., 1979, Meth. Enzym. 62:308; Engval, et al., 1972, Immunol.109:129; Goding, 1976, J. Immunol. Meth. 13:215). The labeled antibodiesof the present invention can be used for in vitro, in vivo, and in situassays to identify cells or tissues which express a specific peptide.

[0157] In another embodiment of the present invention theabove-described antibodies are immobilized on a solid support. Examplesof such solid supports include plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, acrylic resins and such aspolyacrylamide and latex beads. Techniques for coupling antibodies tosuch solid supports are well known in the art (Weir, et al., In:“Handbook of Experimental Immunology,” 4th Ed., Blackwell ScientificPublications, Oxford, England, Chapter 10 (1986); Jacoby, et al., 1974,Meth. Enzym. Vol. 34. Academic Press, N.Y.). The immobilized antibodiesof the present invention can be used for in vitro, in vivo, and in situassays as well as in immunochromatography.

[0158] Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides (Hurby, et al., In:“Application of Synthetic Peptides: Antisense Peptides,” In SyntheticPeptides, A User's Guide, W. H. Freeman, N.Y., pp. 289-307 (1992);Kaspczak, et al., 1989, Biochemistry 28:9230-9238).

[0159] Anti-peptide peptides can be generated in one of two fashions.First, the anti-peptide peptides can be generated by replacing the basicamino acid residues found in the torsin peptide sequence with acidicresidues, while maintaining hydrophobic and uncharged polar groups. Forexample, lysine, arginine, and/or histidine residues are replaced withaspartic acid or glutamic acid and glutamic acid residues are replacedby lysine, arginine or histidine

[0160] In another embodiment, the present invention relates to a methodof detecting a torsin polypeptide in a sample, comprising: contactingthe sample with an above-described antibody (or protein), underconditions such that immunocomplexes form, and detecting the presence ofthe antibody bound to the polypeptide. In detail, the methods compriseincubating a test sample with one or more of the antibodies of thepresent invention and assaying whether the antibody binds to the testsample. Altered levels of torsin in a sample as compared to normallevels can indicate a specific disease.

[0161] In a further embodiment, the present invention relates to amethod of detecting a torsin antibody in a sample, comprising:contacting the sample with an above-described torsin protein, underconditions such that immunocomplexes form, and detecting the presence ofthe protein bound to the antibody or antibody bound to the protein. Indetail, the methods comprise incubating a test sample with one or moreof the proteins of the present invention and assaying whether theantibody binds to the test sample.

[0162] Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention (Chard, In: AnIntroduction to Radioimmunoassay and Related Techniques, ElsevierScience Publishers, Amsterdam, The Netherlands (1986); Bullock, et al.,In: Techniques in Immunocytochemistry, Academic Press, Orlando, Fla.Vol. 1(1982), Vol. 2(1983), Vol. 3(1985); Tijssen, In: Practice andTheory of enzyme Immunoassays: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1985)).

[0163] The immunological assay test samples of the present inventioninclude cells, protein or membrane extracts of cells, or biologicalfluids such as blood, serum, plasma, or urine. The test sample used inthe above-described method will vary based on the assay format, natureof the detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is capable with the systemutilized.

[0164] The claimed invention utilizes several suitable assays which canmeasure dystonia proteins. Suitable assays encompass immunologicalmethods, such as radioimmunoassay, enzyme-linked immunosorbent assays(ELISA), and chemiluminescence assays. Any method known now or developedlater can be used for performing the invention and measuring measuretorsin proteins.

[0165] In several of the preferred embodiments, immunological techniquesdetect torsin proteins levels by means of an anti- dystonia proteinantibody (i.e., one or more antibodies) which includes monoclonal and/orpolyclonal antibodies, and mixtures thereof. For example, theseimmunological techniques can utilize mixtures of polyclonal and/ormonoclonal antibodies, such as a cocktail of murine monoclonal andrabbit polyclonal.

[0166] One of skill in the art can raise anti-torsin antibodies againstan appropriate immunogen, such as isolated and/or recombinant torsinproteins or a portion or fragment thereof (including syntheticmolecules, such as synthetic peptides). In one embodiment, antibodiesare raised against an isolated and/or recombinant torsin proteins or aportion or fragment thereof (e.g., a peptide) or against a host cellwhich expresses recombinant dystonia proteins. In addition, cellsexpressing recombinant torsin proteins, such as transfected cells, canbe used as immunogens or in a screen for antibodies which bind torsinproteins.

[0167] Any suitable technique can prepare the immunizing antigen andproduce polyclonal or monoclonal antibodies. The prior art contains avariety of these methods (Kohler, et al., 1975, Nature. 256:495-497;Kohler, et al., 1976, Eur. J. Immunol. 6:511-519; Milstein, et al.,1977, Nature. 266:550-552; Koprowski, et al., U.S. Pat. No.: 4,172,124;Harlow, et al., In: Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1988)). Generally, fusing asuitable immortal or myeloma cell line, such as SP2/0, with antibodyproducing cells can produce a hybridoma. Animals immunized with theantigen of interest provide the antibody-producing cell, preferablycells from the spleen or lymph nodes. Selective culture conditionsisolate antibody producing hybridoma cells while limiting dilutiontechniques produce well established art recognized assays such as ELISA,RIA and Western blotting can be used to select antibody producing cellswith the desired specificity.

[0168] Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice which are capable of producing a fullrepertoire of human antibodies (Jakobovits, et al., 1993, Proc. Natl.Acad. Sci. USA 90:2551-2555; Jakobovits, et al., 1993, Nature.362:255-258; Lonbert, et al., U.S. Pat. No.: 5,545,806; Surani, et al.,U.S. Pat. No.: 5,545,807).

[0169] According to the method, an assay can determine the level orconcentration of torsin protein in a biological sample. In determiningthe amounts of torsin protein, an assay includes combining the sample tobe tested with an antibody having specificity for torsin proteins, underconditions suitable for formation of a complex between antibody andtorsin protein, and detecting or measuring (directly or indirectly) theformation of a complex. The sample can be obtained and prepared by amethod suitable for the particular sample (e.g., whole blood, tissueextracts, serum) and assay format selected. For example, suitablemethods for whole blood collection are venipuncture or obtaining bloodfrom an indwelling arterial line. The container to collect the blood cancontain an anti-coagulant such as CACD-A, heparin, or EDTA. Methods ofcombining sample and antibody, and methods of detecting complexformation are also selected to be compatible with the assay format.Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels; or indirectly detected usinglabels such as enzyme labels and other antigenic or specific bindingpartners like biotin and colloidal gold. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, CY5, APC,chemiluminescent labels such as luciferase, radioisotope labels such as.sup.32p, .sup.1251, .sup.131I, enzyme labels such as horseradishperoxidase, and alkaline phosphatase, 0-galactosidase, biotin, avidin,spin labels and the like. The detection of antibodies in a complex canalso be done immunologically with a second antibody which is thendetected. Conventional methods or other suitable methods can directly orindirectly label an antibody.

[0170] In another embodiment of the present invention, a kit is providedfor diagnosing the presence or absence of a torsin protein; or thelikelihood of developing a dystonia in a mammal which contains all thenecessary reagents to carry out the previously described methods ofdetection.

[0171] For example, the kit can comprise a first container meanscontaining an above described antibody, and a second container meanscontaining a conjugate comprising a binding partner of the antibody anda label.

[0172] The kit can also comprise a first container means containing anabove described protein, and preferably and a second container meanscontaining a conjugate comprising a binding partner of the protein and alabel. More specifically, a diagnostic kit comprises torsin protein asdescribed above, to detect antibodies in the serum of potentiallyinfected animals or humans.

[0173] In another preferred embodiment, the kit further comprises one ormore other containers comprising one or more of the following: washreagents and reagents capable of detecting the presence of boundantibodies. Examples of detection reagents include, but are not limitedto, labeled secondary antibodies, or in the alternative, if the primaryantibody is labeled, the chromophoric, enzymatic, or antibody bindingreagents which are capable of reacting with the labeled antibody. Thecompartmentalized kit can be as described above for nucleic acid probekits. The kit can be, for example, a RIA kit or an ELISA kit.

[0174] One skilled in the art will readily recognize that the antibodiesdescribed in the present invention can readily be incorporated into oneof the established kit formats which are well known in the art.

[0175] It is to be understood that although the following discussion isspecifically directed to human patients, the teachings are alsoapplicable to any animal that expresses a torsin protein. The term“mammalian,” as defined herein, refers to any vertebrate animal,including monotremes, marsupials and placental, that suckle their youngand either give birth to living young (eutherian or placental mammals)or are egg-laying (metatherian or non-placental mammals). Examples ofmammalian species include primates (e.g., humans, monkeys, chimpanzees,baboons), rodents (e.g., rats, mice, guinea pigs, hamsters) andruminants (e.g., cows, horses).

[0176] The diagnostic and screening methods of the present inventionencompass detecting the presence, or absence of, a mutation in a genewherein the mutation in the gene results in a neuronal disease in ahuman. For example, the diagnostic and screening methods of the presentinvention are especially useful for diagnosing the presence or absenceof a mutation or polymorphism in a neuronal gene in a human patient,suspected of being at risk for developing a disease associated with analtered expression level of torsin based on family history, or a patientin which it is desired to diagnose a torsin-related disease.

[0177] Preferably, nucleic acid diagnosis is used as a means ofdifferential diagnosis of various forms of a torsion dystonia such asearly-onset generalized dystonia; late-onset generalized dystonia; orany form of genetic, environmental, primary or secondary dystonia. Thisinformation is then used in genetic counseling and in classifyingpatients with respect to individualized therapeutic strategies.

[0178] According to the invention, presymptomatic screening of anindividual in need of such screening is now possible using DNA encodingthe torsin protein of the invention. The screening method of theinvention allows a presymptomatic diagnosis, including prenataldiagnosis, of the presence of a missing or aberrant torsin gene inindividuals, and thus an opinion concerning the likelihood that suchindividual would develop or has developed a torsin-associated disease.This is especially valuable for the identification of carriers ofaltered or missing torsin genes, for example, from individuals with afamily history of a torsin-associated disease. Early diagnosis is alsodesired to maximize appropriate timely intervention.

[0179] Identification of gene carriers prior to onset of symptoms allowsevaluation of genetic and environmental factors that trigger onset ofsymptoms. Modifying genetic factors could include polymorphic variationsin torsin proteins (specifically, torsin proteins) or mutations inrelated or associated proteins; environmental factors include sensoryoverload to the part of body subserved by susceptible neurons, such asthat caused by overuse or trauma (Gasser, T., et al., 1996, Mov Disord.11:163 -166); high body temperature; or exposure to toxic agents.

[0180] In one embodiment of the diagnostic method of screening, a testsample comprising a bodily fluid (e.g., blood, saliva, amniotic fluid)or a tissue (e.g., neuronal, chorionic villous) sample would be takenfrom such individual and screened for (1) the presence or absence of the“normal” torsin gene; (2) the presence or absence of torsin mRNA and/or(3) the presence or absence of torsin protein. The normal human gene canbe characterized based upon, for example, detection of restrictiondigestion patterns in “normal” versus the patients DNA, including RFLP,PCR, Southern blot, Northern blot and nucleic acid sequence analysis,using DNA probes prepared against the torsin sequence (or a functionalfragment thereof) taught in the invention. In one embodiment the torsinsequence is a torsin sequence (SEQ ID NOS: 1, 3, 5, 7, and 9). Inanother embodiment the presence or absence of three nucleotides isindicative of a negative or positive diagnosis, respectively, of atorsion dystonia. Similarly, torsin mRNA can be characterized andcompared to normal torsin mRNA (a) levels and/or (b) size as found in ahuman population not at risk of developing torsin-associated diseaseusing similar probes. Additionally or alternatively, nucleic acids canbe sequenced to determine the presence or absence of a “normal” torsingene. Nucleic acids can be DNA (e.g., cDNA or genomic DNA) or RNA.

[0181] Lastly, torsin protein can be (a) detected and/or (b) quantitatedusing a biological assay for torsin activity or using an immunologicalassay and torsin antibodies. When assaying torsin protein, theimmunological assay is preferred for its speed. In one embodiment of theinvention the torsin protein sequence (SEQ ID NOS: 2, 4, 6, 8, and 10)or a protein encoded by SEQ ID NOS: 1, 3, 5, 7, and 9. An (1) aberranttorsin DNA size pattern, and/or (2) aberrant torsin mRNA sizes or levelsand/or (3) aberrant torsin protein levels would indicate that thepatient is at risk for developing a torsin-associated disease.

[0182] Mutations associated with a dystonia disorder include anymutation in a dystonia gene, such as tor-2. The mutations can be thedeletion or addition of at least one nucleotide in the coding ornoncoding region, of the tor-2 gene which result in a change in a singleamino acid or in a frame shift mutation.

[0183] In one method of diagnosing the presence or absence of a dystoniadisorder, hybridization methods, such as Southern analysis, are used(Ausubel, et al., In: Current Protocols in Molecular Biology, John Wiley& Sons, (1998)). Test samples suitable for use in the present inventionencompass any sample containing nucleic acids, either DNA or RNA. Forexample, a test sample of genomic DNA is obtained from a human suspectedof having (or carrying a defect for) the dystonia disorder. The testsample can be from any source which contains genomic DNA, such as abodily fluid or tissue sample. In one embodiment, the test sample of DNAis obtained from bodily fluids such as blood, saliva, semen, vaginalsecretions, cerebrospinal and amniotic bodily fluid samples. In anotherembodiment, the test sample of DNA is obtained from tissue such aschorionic villous, neuronal, epithelial, muscular and connective tissue.DNA can be isolated from the test samples using standard, art-recognizedprotocols (Breakefield, X. O., et al., 1986, J. Neurogenetics.3:159-175). The DNA sample is examined to determine whether a mutationassociated with a dystonia disorder is present or absent. The presenceor absence of a mutation or a polymorphism is indicated by hybridizationwith a neuronal gene, such as the tor-2 gene, in the genomic DNA to anucleic acid probe. A nucleic acid probe is a nucleotide sequence of aneuronal gene. Additionally or alternatively, RNA encoded by such aprobe can also be used to diagnose the presence or absence of a dystoniadisorder by hybridization, a hybridization sample is formed bycontacting the test sample containing a dystonia gene, such as tor-2,with a nucleic acid probe. The hybridization sample is maintained underconditions which are sufficient to allow specific hybridization of thenucleic acid probe to the dystonia gene of interest. Hybridization canbe carried out as discussed previously above.

[0184] In another embodiment of the invention, deletion analysis byrestriction digestion can be used to detect a deletion in a dystoniagene, such as the tor-2 gene, if the deletion in the gene results in thecreation or elimination of a restriction site. For example, a testsample containing genomic DNA is obtained from the human. Afterdigestion of the genomic DNA with an appropriate restriction enzyme, DNAfragments are separated using standard methods, and contacted with aprobe specific for the a torsin gene or cDNA. The digestion pattern ofthe DNA fragments indicates the presence or absence of the mutationassociated with a dystonia disorder. Alternatively, polymerase chainreaction (PCR) can be used to amplify the dystonia gene of interest,such as tor-2, (and, if necessary, the flanking sequences) in a testsample of genomic DNA from the human. Direct mutation analysis byrestriction digestion or nucleotide sequencing is then conducted. Thedigestion pattern of the relevant DNA fragment indicates the presence orabsence of the mutation associated with the dystonia disorder.

[0185] Allele-specific oligonucleotides can also be used to detect thepresence or absence of a neuronal disease by detecting a deletion or apolymorphism associated with a particular disease by PCR amplificationof a nucleic acid sample from a human with allele-specificoligonucleotide probes. An “allele-specific oligonucleotide” (alsoreferred to herein as an “allele-specific oligonucleotide probe”) is anoligonucleotide of approximately 10-300 base pairs, that specificallyhybridizes to a dystonia gene, such as tor-2, (or gene fragment) thatcontains a particular mutation, such as a deletion of three nucleotides.An allele-specific oligonucleotide probe that is specific for particularmutation in, for example, the tor-2 gene, can be prepared, usingstandard methods (Ausubel, et al., In: Current Protocols in MolecularBiology, John Wiley & Sons, (1998)).

[0186] To identify mutations in the tor-2 gene associated with torsiondystonia, or any other neuronal disease a test sample of DNA is obtainedfrom the human. PCR can be used to amplify all or a fragment of thetor-2 gene, and its flanking sequences. PCR primers comprise anysequence of a neuronal gene. The PCR products containing the amplifiedneuronal gene, for example a tor-2 gene (or fragment of the gene), areseparated by gel electrophoresis using standard methods (Ausubel, etal., In: Current Protocols in Molecular Biology, John Wiley & Sons,(1998)), and fragments visualized using art-recognized, well-establishedtechniques such as fluorescent imaging when fluorescently labeledprimers are used. The presence or absence of specific DNA fragmentsindicative of the presence or absence of a mutation or a polymorphism ina neuronal gene are then detected. For example, the presence of twoalleles of a specific molecular size is indicative of the absence of atorsion dystonia; whereas the absence of one of these alleles isindicative of a torsion dystonia. The samples obtained from humans andevaluated by the methods described herein will be compared to standardsamples that do and do not contain the particular mutations orpolymorphism which are characteristic of the particular neuronaldisorder.

[0187] Prenatal diagnosis can be performed when desired, using any knownmethod to obtain fetal cells, including amniocentesis, chorionic villoussampling (CVS), and fetoscopy. Prenatal chromosome analysis can be usedto determine if the portion of the chromosome possessing the normaltorsin gene is present in a heterozygous state In the method of treatinga torsin-associated disease in a patient in need of such treatment,functional torsin DNA can be provided to the cells of such patient in amanner and amount that permits the expression of the torsin proteinprovided by such gene, for a time and in a quantity sufficient to treatsuch patient. Many vector systems are known in the art to provide suchdelivery to human patients in need of a gene or protein missing from thecell. For example, retrovirus systems can be used, especially modifiedretrovirus systems and especially herpes simplex virus systems(Breakefield, X. O., et al., 1991, New Biologist. 3:203-218; Huang, Q.,et al., 1992, Experimental Neurology. 115:303-316; WO93/03743;WO90/09441). Delivery of a DNA sequence encoding a functional torsinprotein will effectively replace the missing or mutated torsin gene ofthe invention In another embodiment of this invention, the torsin geneis expressed as a recombinant gene in a cell, so that the cells can betransplanted into a mammal, preferably a human in need of gene therapy.To provide gene therapy to an individual, a genetic sequence whichencodes for all or part of the torsin gene is inserted into a vector andintroduced into a host cell. Examples of diseases that can be suitablefor gene therapy include, but are not limited to, neurodegenerativediseases or disorders, primary dystonia (preferably, generalizeddystonia and torsion dystonia).

[0188] Gene therapy methods can be used to transfer the torsin codingsequence of the invention to a patient (Chattedee and Wong, 1996, Curr.Top. Microbiol. Immunol. 218:61-73; Zhang, 1996, J. Mol. Med.74:191-204; Schmidt-Wolf and Schmidt-Wolf, 1995, J. Hematotherapy.4:551-561; Shaughnessy, et al., 1996, Seminars in Oncology. 23:159-171;Dunbar, 1996, Annu. Rev. Med. 47:11-20

[0189] Examples of vectors that may be used in gene therapy include, butare not limited to, defective retroviral, adenoviral, or other viralvectors (Mulligan, R. C., 1993, Science. 260:926-932). The means bywhich the vector carrying the gene can be introduced into the cellinclude but is not limited to, microinjection, electroporation,transduction, or transfection using DEAE-Dextran, lipofection, calciumphosphate or other procedures known to one skilled in the art (Sambrook,J., Fritsch, E. F., and Maniatis, T., 1989, In: Molecular Cloning. ALaboratory Manual., Cold Spring Harbor Laboratory Press, Cold SpringHarbor).

[0190] The ability of antagonists and agonists of torsin to interfere orenhance the activity of torsin can be evaluated with cells containingtorsin. An assay for torsin activity in cells can be used to determinethe functionality of the torsin protein in the presence of an agentwhich may act as antagonist or agonist, and thus, agents that interfereor enhance the activity of torsin are identified

[0191] The agents screened in the assays can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. These agents can be selected and screened at random, by arational selection or by design using, for example, protein or ligandmodeling techniques (preferably, computer modeling).

[0192] For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to or stimulate/block the activity ofthe torsin protein.

[0193] Alternatively, agents may be rationally selected or designed. Asused herein, an agent is said to be “rationally selected or designed”when the agent is chosen based on the configuration of the torsinprotein.

[0194] In one embodiment, the present invention relates to a method ofscreening for an antagonist or agonist which stimulates or blocks theactivity of torsin comprising incubating a cell expressing torsin withan agent to be tested; and assaying the cell for the activity of thetorsin protein by measuring the agents effect on ATP binding of torsin.Any cell may be used in the above assay so long as it expresses afunctional form of torsin and the torsin activity can be measured. Thepreferred expression cells are eukaryotic cells or organisms. Such cellscan be modified to contain DNA sequences encoding torsin using routineprocedures known in the art. Alternatively, one skilled in the art canintroduce mRNA encoding the torsin protein directly into the cell.

[0195] In another embodiment, the present invention relates to a screenfor pharmaceuticals (e.g., drugs) which can counteract the expression ofa mutant torsin protein. Preferably, a neuronal culture is used for theoverexpression of the mutant form of torsin proteins using the vectortechnology described herein. Changes in neuronal morphology and proteindistribution is assessed and a means of quantification is used. Thisbioassay is then used as a screen for drugs which can ameliorate thephenotype. Using torsin ligands (including antagonists and agonists asdescribed above) the present invention further provides a method formodulating the activity of the torsin protein in a cell. In general,agents (antagonists and agonists) which have been identified to block orstimulate the activity of torsin can be formulated so that the agent canbe contacted with a cell expressing a torsin protein in vivo. Thecontacting of such a cell with such an agent results in the in vivomodulation of the activity of the torsin proteins. So long as aformulation barrier or toxicity barrier does not exist, agentsidentified in the assays described above will be effective for in vivouse.

[0196] In another embodiment, the present invention relates to a methodof administering torsin or a torsin ligand (including torsin antagonistsand agonists) to an animal (preferably, a mammal (specifically, ahuman)) in an amount sufficient to effect an altered level of torsin inthe animal. The administered torsin or torsin ligand could specificallyeffect torsin associated functions. Further, since torsin is expressedin brain tissue, administration of torsin or torsin ligand could be usedto alter torsin levels in the brain.

[0197] One skilled in the art will appreciate that the amounts to beadministered for any particular treatment protocol can readily bedetermined. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of disease in the patient, counter indications, if any, andother such variables, to be adjusted by the individual physician. Thedosages used in the present invention to provide immunostimulationinclude from about 0.1 μg to about 500 μg, which includes, 0.5, 1.0,1.5, 2.0, 5.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, and 450 μg, inclusiveof all ranges and subranges there between. Such amount may beadministered as a single dosage or may be administered according to aregimen, including subsequent booster doses, whereby it is effective,e.g., the compositions of the present invention can be administered onetime or serially over the course of a period of days, weeks, monthsand/or years.

[0198] Also, the dosage form such as injectable preparations (solutions,suspensions, emulsions, solids to be dissolved when used, etc.),tablets, capsules, granules, powders, liquids, liposome inclusions,ointments, gels, external powders, sprays, inhalating powders, eyedrops, eye ointments, suppositories, pessaries, and the like can be usedappropriately depending on the administration method, and the peptide ofthe present invention can be accordingly formulated. Pharmaceuticalformulations are generally known in the art, and are described, forexample, in Chapter 25.2 of Comprehensive Medicinal Chemistry, Volume 5,Editor Hansch et al, Pergamon Press 1990.

[0199] Torsin or torsin ligand can be administered parenterally byinjection or by gradual perfusion over time. It can be administeredintravenously, intraperitoneally, intramuscularly, or subcutaneously.

[0200] Preparations for parenteral administration include sterile oraqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives can also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like (Remington's Pharmaceutical Science, 16th ed., Eds.:Osol, A., Ed., Mack, Easton Pa. (1980)).

[0201] In another embodiment, the present invention relates to apharmaceutical composition comprising torsin or torsin ligand in anamount sufficient to alter is torsin associated activity, and apharmaceutically acceptable diluent, carrier, or excipient. Appropriateconcentrations and dosage unit sizes can be readily determined by oneskilled in the art as described above (Remington's PharmaceuticalSciences, 16th ed., Eds.: Osol, A., Ed., Mack, Easton Pa. (1980); WO91/19008).

[0202] The pharmaceutically acceptable carrier which can be used in thepresent invention includes, but is not limited to, an excipient, abinder, a lubricant, a colorant, a disintegrant, a buffer, an isotonicagent, a preservative, an anesthetic, and the like which are commonlyused in a medical field.

[0203] The non-human animals of the invention comprise any animal havinga transgenic interruption or alteration of the endogenous gene(s)(knock-out animals) and/or into the genome of which has been introducedone or more transgenes that direct the expression of human torsin.

[0204] Such non-human animals include vertebrates such as rodents,non-human primates, sheep, dog, cow, amphibians, reptiles, etc.Preferred non-human animals are selected from non-human mammalianspecies of animals, most preferably, animals from the rodent familyincluding rats and mice, most preferably mice.

[0205] The transgenic animals of the invention are animals into whichhas been introduced by nonnatural means (i. e., by human manipulation),one or more genes that do not occur naturally in the animal, e.g.,foreign genes, genetically engineered endogenous genes, etc. Thenon-naturally introduced genes, known as transgenes, may be from thesame or a different species as the animal but not naturally found in theanimal in the configuration and/or at the chromosomal locus conferred bythe transgene.

[0206] Transgenes may comprise foreign DNA sequences, i.e., sequencesnot normally found in the genome of the host animal. Alternatively oradditionally, transgenes may comprise endogenous DNA sequences that areabnormal in that they have been rearranged or mutated in vitro in orderto alter the normal in vivo pattern of expression of the gene, or toalter or eliminate the biological activity of an endogenous gene productencoded by the gene (Watson, J. D., et al., In: Recombinant DNA, 2d Ed.,W. H. Freeman & Co., New York (1992), pg. 255-272; Gordon, J. W., 1989,Intl. Rev. Cytol. 115:171-229; Jaenisch, R., 1989, Science.240:1468-1474; Rossant, J., 1990, Neuron. 2:323-334).

[0207] The transgenic non-human animals of the invention are produced byintroducing transgenes into the germline of the non-human animal.Embryonic target cells at various developmental stages are used tointroduce the transgenes of the invention. Different methods are useddepending on the stage of development of the embryonic target cell(sMicroinjection of zygotes is the preferred method for incorporatingtransgenes into animal genome in the course of practicing the invention.A zygote, a fertilized ovum that has not undergone pronuclei fusion orsubsequent cell division, is the preferred target cell formicroinjection of transgenic DNA sequences. The murine male pronucleusreaches a size of approximately 20 micrometers in diameter, a featurewhich allows for the reproducible injection of 1-2 pL of a solutioncontaining transgenic DNA sequences. The use of a zygote forintroduction of transgenes has the advantage that, in most cases, theinjected transgenic DNA sequences will be incorporated into the hostanimal's genome before the first cell division (Brinster, et al., 1985,Proc. Natl. Acad. Sci. USA 82:4438-4442). As a consequence, all cells ofthe resultant transgenic animals (founder animals) stably carry anincorporated transgene at a particular genetic locus, referred to as atransgenic allele. The transgenic allele demonstrates Mendelianinheritance: half of the offspring resulting from the cross of atransgenic animal with a non-transgenic animal will inherit thetransgenic allele, in accordance with Mendel's rules of randomassortment.

[0208] Viral integration can also be used to introduce the transgenes ofthe invention into an animal. The developing embryos are cultured invitro to the developmental stage known as a blastocyst. At this time,the blastomeres may be infected with appropriate retroviruses (Jaenisch,R., 1976, Proc. Natl. Acad. Sci. USA 73:1260-1264). Infection of theblastomeres is enhanced by enzymatic removal of the zona pellucida(Hogan, et al., In: Manipulating the Mouse Embryo, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1986)). Transgenes are introduced viaviral vectors which are typically replication-defective but which remaincompetent for integration of viral-associated DNA sequences, includingtransgenic DNA sequences linked to such viral sequences, into the hostanimal's genome (Jahner, et al., 1985, Proc. Natl. Acad. Sci. USA82:6927-6931; van der Putten, et al., 1985, Proc. Natl. Acad. Sci. USA82:6148-6152). Transfection is easily and efficiently obtained byculture of blastomeres on a mono-layer of cells producing thetransgene-containing viral vector (van der Putten, et al., 1985, Proc.Natl. Acad. Sci. USA 82:6148-6152; Stewart, et al., 1987, EMBO J.6:383-388). Alternatively, infection may be performed at a later stage,such as a blastocoele (Jahner, D., et al., 1982, Nature. 298:623-628).In any event, most transgenic founder animals produced by viralintegration will be mosaics for the transgenic allele; that is, thetransgene is incorporated into only a subset of all the cells that formthe transgenic founder animal. Moreover, multiple viral integrationevents may occur in a single founder animal, generating multipletransgenic alleles which will segregate in future generations ofoffspring. Introduction of transgenes into germline cells by this methodis possible but probably occurs at a low frequency (Jahner, D., et al.,1982, Nature. 298:623-628). However, once a transgene has beenintroduced into germline cells by this method, offspring may be producedin which the transgenic allele is present in all of the animal's cells,i.e., in both somatic and germline cells.

[0209] Embryonic stem (ES) cells can also serve as target cells forintroduction of the transgenes of the invention into animals. ES cellsare obtained from pre-implantation embryos that are cultured in vitro(Evans, M. J., et al., 1981, Nature. 292:154-156; Bradley, M. O., etal., 1984, Nature. 309:255-258; Gossler, et al., 1986, Proc. Natl. Acad.Sci. USA 83:9065-9069; Robertson, E. J., et al., 1986, Nature.322:445-448; Robertson, E. J., In: Teratocarcinomas and Embryonic StemCells: A Practical, Approach, Ed.: Robertson, E. J., IRL Press, Oxford(1987), pg. 71-112). ES cells, which are commercially available (from,e.g., Genome Systems, Inc., St. Louis, Mo.), can be transformed with oneor more transgenes by established methods (Lovell-Badge, R. H., In:Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Ed.:Robertson, E. J., IRL Press, Oxford (1987), pg. 153-182). Transformed EScells can be combined with an animal blastocyst, after which the EScells colonize the embryo and contribute to the germline of theresulting animal, which is a chimera composed of cells derived from twoor more animals (Jaenisch, R., 1988, Science. 240:1468-1474; Bradley,A., In: Teratocarcinomas and Embryonic Stem Cells. A Practical Approach,Ed.: Robertson, E. J., IRL Press, Oxford (1987), pg. 113-151). Again,once a transgene has been introduced into germline cells by this method,offspring may be produced in which the transgenic allele is present inall of the animal's cells, i.e., in both somatic and germline cells.

[0210] However it occurs, the initial introduction of a transgene is anon-Mendelian event. However, the transgenes of the invention may bestably integrated into germline cells and transmitted to offspring ofthe transgenic animal as Mendelian loci. Other transgenic techniquesresult in mosaic transgenic animals, in which some cells carry thetransgenes and other cells do not. In mosaic transgenic animals in whichgerm line cells do not carry the transgenes, transmission of thetransgenes to offspring does not occur. Nevertheless, mosaic transgenicanimals are capable of demonstrating phenotypes associated with thetransgenes.

[0211] Transgenes may be introduced into non-human animals in order toprovide animal models for human diseases. Transgenes that result in suchanimal models include, e.g., transgenes that encode mutant gene productsassociated with an inborn error of metabolism in a human genetic diseaseand transgenes that encode a human factor required to confersusceptibility to a human pathogen (i.e., a bacterium, virus, or otherpathogenic microorganism; Leder, et al., U.S. Pat. No. 5,175,383; Kindt,et al., U.S. Pat. No. 5,183,949; Small, et al., 1986, Cell. 46:13-18;Hooper, et al., 1987, Nature. 326:292-295; Stacey, et al., 1988, Nature.332:131-136; Windle, et al., 1990, Nature. 343:665-669; Katz, et al.,1993, Cell. 74:1089-1100). Transgenically introduced mutations can giverise to null (“knock-out”) alleles in which a DNA sequence encoding aselectable and/or detectable marker is substituted for a geneticsequence normally endogenous to a non-human animal. Resultant transgenicnon-human animals that are predisposed to a disease, or in which thetransgene causes a disease, may be used to identify compositions thatinduce the disease and to evaluate the pathogenic potential ofcompositions known or suspected to induce the disease (Bems, A. J. M.,U.S. Pat. No. 5,174,986), or to evaluate compositions which may be usedto treat the disease or ameliorate the symptoms thereof (Scott, et al.,WO 94/12627).

[0212] Offspring that have inherited the transgenes of the invention aredistinguished from litter mates that have not inherited transgenes byanalysis of genetic material from the offspring for the presence ofbiomolecules that comprise unique sequences corresponding to sequencesof, or encoded by, the transgenes of the invention. For example,biological fluids that contain polypeptides uniquely encoded by theselectable marker of the transgenes of the invention may beimmunoassayed for the presence of the polypeptides. A more simple andreliable means of identifying transgenic offspring comprises obtaining atissue sample from an extremity of an animal, e.g., a tail, andanalyzing the sample for the presence of nucleic acid sequencescorresponding to the DNA sequence of a unique portion or portions of thetransgenes of the invention, such as the selectable marker thereof. Thepresence of such nucleic acid sequences may be determined by, e.g.,Southern blot analysis with DNA sequences corresponding to uniqueportions of the transgene, analysis of the products of PCR reactionsusing DNA sequences in a sample as substrates and oligonucleotidesderived from the transgene's DNA sequence, etc.

[0213] In another embodiment, the present invention relates to arecombinant DNA molecule comprising an HSV-1 amplicon and at least oneabove-described torsin nucleic acid molecule.

[0214] Several features make HSV-1 an ideal candidate for vectordevelopment: (i) HSV-1 is essentially pantropic and can infect bothdividing and non-dividing cells, such as neurons and hepatocytes; (ii)the HSV-1 genome can remain in neurons for long periods with at leastsome transcriptional activity; and (iii) the HSV-1 genome encodes morethan 75 genes of which 38 are dispensable (nonessential) for viralreplication in cell culture (Ward, P. L. and Roizinan, B., 1994, TrendsGenet. 10:267-274). This offers the opportunity to replace large partsof the genome with foreign DNA, including one or more therapeutic genesof interest.

[0215] The technology to construct recombinant HSV-I vectors wasdeveloped more than a decade ago (Mocarski, E. S., et al., 1980, Cell.22:243-255; Post, L. E. and Reizman, B., 1981, Cell. 25:2227-2232;Roizman, B. and F. J. Jenkins, 1985, Science. 229:1208-1214). With thegoal to create a prototype HSV-1/HSV-2 recombinant vaccine, the HSV-1genome was deleted in certain domains in order to eliminate some lociresponsible for neurovirulence, such as the viral thymidine kinase gene,and to create space for the insertion of a DNA fragment encoding theherpes simplex virus type 2 (HSV-2) glycoproteins D, G, and I (Meignier,B., et al., 1988, J. Inf. Dis. 158:602-614). Currently, recombinantherpes virus vectors are being evaluated in numerous protocols primarilyfor gene therapy of neurodegenerative diseases and brain tumors(Breakefield, X. O., et al, In: Cancer Gene Therapeutics, (1995), pp.41-56; Glorioso, J. C., et al., “Herpes simplex virus as a gene-deliveryvector for the central nervous system,” In: Viral vectors: Gene therapyand neuroscience applications, Eds.: Kaplitt, M. G. and Loewy, A. D.,Academic Press, NY (1995), pp. 1-23).

[0216] The development of a second type of HSV-1 vector, the so-calledHSV-1 “amplicon” vector, was based on the characterization of naturallyoccurring defective HSV-I genomes (Frenkel, N., et al., 1976, J. Virol.20:527-531). Amplicons carry three types of genetic elements: (i)prokaryotic sequences for propagation of plasmid DNA in bacteria,including an E. coli origin of DNA replication and an antibioticresistance gene; (ii) sequences from HSV-1, including an ori and a pacsignal to support replication and packaging into HSV-1 particles inmammalian cells in the presence of helper virus functions; and (iii) atranscription unit with one or more genes of interest (Ho, D. Y., 1994,Meth. Cell. Biol. 43:191-210) defective viruses and development of theamplicon system (Viral vectors: Gene therapy and neuroscienceapplications, Eds.: Kaplitt, M. G., and Loewy, A. D., Academic Press, NY(1995), pp. 25-42).

[0217] In another embodiment, the present invention relates to the useof the above-described amplicon vectors for transfer of a torsin nucleicacid molecule into neurons HSV-1 has several biological properties thatfacilitate its use as a gene transfer vector into the CNS. Theseinclude: (i) a large transgene capacity (theoretically up to 150 kb),(ii) tropism for the CNS in vivo, (iii) nuclear localization in dividingas well as non-dividing cells, (iv) a large host cell range in tissueculture, (v) the availability of a panel of neuroattenuated andreplication incompetent mutants, and (vi) the possibility to producerelatively high virus titers. Another important property of the HSV-1derived vector systems for the CNS is the ability of these virions to betransported retrogradely along axons. After fusion with the cellmembrane, the virus capsid and associated tegument proteins are releasedinto the cytoplasm. These capsids associate with the dynein complexwhich mediates energy dependent retrograde transport to the cell nucleusalong microtubules (Topp, K. S., et al, 1994, J. Neurosci. 14:318-325).Replication-incompetent, recombinant and amplicon HSV-1 vectorsexpressing the lacZ gene have been used to determine the localizationand spread of vectors after injection. After single injections into manyareas, including caudate nucleus, dentate gyrus and cerebellar cortex,the distribution of .beta.-galactosidase-positive cells was determined(Chiocca, E. A., et al., 1990, N. Biol. 2:739-746; Fink, D. J., et al.,1992, Hum. Gene Ther. 3:11-19; Huang, Q., et al., 1992, Exp. Neurol.115:303-316; Wood, M., et al., 1994, Exp. Neurol. 130:127-140). Neuronsand glia were transduced at the site of injection, and activity was alsodetected at distant secondary brain areas, in neurons that make afferentconnections with the cells in the primary injection site. The retrogradetransport to secondary sites is selective to neuroanatomic pathways,suggesting trans-synaptic travel of the virus capsids. Retrogradetransport of an amplicon vector has been demonstrated after striatalinjections in both the substantia nigra pars compacta and the locuscoeruleus (Jin, B. K., et al., 1996, Hum. Gene Ther. 7:2015-2024). Theability of HSV-1 to travel by retrograde transport to neurons inafferent pathways suggests that the delivery of genes by these vectorscan be spread beyond the original injection site to other regions ofneuroanatomic importance. The original report of amplicon-mediated genedelivery to neurons used primary cells in culture (Geller, A. I. andBreakefield, X. O. 1988, Science 241:1667-1669). Amplicon vectors havebeen used to study neuronal physiology, for example effects ofexpression of GAP43 or the low affinity nerve growth factor (NGF)receptor on morphology and growth of neuronal cells (Neve, R. L., etal., 1991, Mol. Neurobiol. 5:131-141; Battleman, D., et al., 1993, J.Neurosci. 13:941-951). Amplicons can direct rapid and stable transgeneexpression in hippocampal slice cultures (Bahr, B., et al., 1994, Mol.Brain Res. 26:277-285), and this has been used to model both kainatereceptor-mediated toxicity (Bergold, P. J., et al., 1993, Proc. NatlAcad. Sci. USA 90:6165-6169) and glucose transporter-mediated protectionof neurons (Ho, D. Y., et al., 1995, J. Neurochem. 65:842-850). In vivo,amplicons have been used to deliver a number of candidate therapeuticgenes in different models of CNS diseases. For example, expression ofthe glucose transporter protects neurons in an induced seizure model((Ho, D. Y., et al., 1995, J. Neurochem. 65:842-850; Lawrence, M. S., etal., 1995, Proc. Natl. Acad. Sci. USA 92:7247-7251; Lawrence, M. S., etal., 1996, Blood Flow Metab. 16:181-185), bcl-2 rescues neurons fromfocal ischemia (Linnik, M. D., et al., 1995, Stroke 26:1670-1674), andexpression of TH mediates behavioral changes in parkinsonian rats(During, M. J., et al., 1994, Science 266:1399-1403). Thus, ampliconshave proven effective for functional expression of many transgenes inthe CNS Amplicons have recently been used to generate mouse somaticmosaics, in which the expression of a host gene is activated in aspatial and developmentally regulated fashion. Transgenic mice wereengineered with a germline transmitted NGF gene that contained aninactivating insertional element between the promoter and transcriptflanked by the loxP sites. The somatic delivery of cre recombinase by anamplicon vector successfully activated the expression of NGF in theseanimals (Brooks, A. I., et al., 1997, Nat. Biotech. 15:57-62). Theability to express genes in specific cells at various points indevelopment will have broad applications, especially for genes for whichgermline deletion (“knockouts”) are conditional lethal mutants.

[0218] Traditionally, the stability of transgene expression aftertransduction, and the cytopathic effect of the helper virus were thelimiting features of amplicon mediated gene delivery into cells of theCNS. Recent advancements have largely addressed these constraints.Several promoter elements, such as preproenkephalin and tyrosinehydroxylase, can drive long-term transgene expression from ampliconvectors when upstream regulatory sequences are included (Kaplitt, M. G.,et al., 1994, Proc. Natl. Acad. Sci. USA 91:8979-8983; Jin, B. K., etal., 1996, Hum. Gene Ther. 7:2015-2024). The development of hybridamplicons containing non-HSV genetic elements that can potentiallyintegrate in a site directed manner (Johnston, K. M., et al., 1997, Hum.Gene Ther. 8:359-370), or form stable replicating episomes (Wang, S. andVos, J., 1996, J. Virol. 70:8422-8430), should maintain the-introducedtransgene in a emetically stable configuration. Finally, the developmentof a packaging system devoid of contaminating helper virus (Fraefel, C.,et al., 1996, J. Virol. 70:7190-7197) has significantly reduced thecytopathic effects of amplicon vectors in culture and in vivo. Theeasily manipulated plasmid-based amplicon, and the helper virus-freepackaging system allows the construction of a virtually synthetic vectorwhich retains the biological advantages of HSV-1, but reduces the risksassociated with virus-based gene therapy.

[0219] In another embodiment, the present invention relates to the useof the above-described amplicon vectors for transfer of a torsin nucleicacid molecule into hepatocytes. As discussed in the previous section,HSV-I amplicon vectors have been extensively evaluated for gene transferinto cells of the nervous system. However, amplicon vectors can also bean efficient means of gene delivery to other tissues, such as the liver.Certain hereditary liver disorders can be treated by enzyme/proteinreplacement or by liver transplantation. However, protein infusion canonly temporarily restore the deficiency and is not effective for manyintracellular proteins. Liver transplantation is limited by donor organavailability and the need for immunosuppression for the lifetime of thepatient. Thus, gene transfer to the liver is highly desirable, andconsequently, various virus vector systems, including adenovirus vectors(Stratford-Perricaudet, L. D., et al., 1990, Hum. Gene Ther. 1:241-256;Jaffe, A. H., et al., 1992, Nat. Genet. 1:372-378; Li, Q., et al., 1993,Hum. Gene Ther. 4:403-409; Herz, J. and Gerard, R. D., 1993, Proc. Natl.Acad. Sci. USA 90:2812-2816), retrovirus vectors (Hafenrichter, D. G.,et al., 1994, Blood 84:3394-3404), baculovirus vectors (Boyce, F. M. andBucher, N. R. L., 1996, Proc. Natl. Acad. Sci. USA 93:2348-2352; Sandig,V., et al., 1996, Hum. Gene Ther. 7:1937-1945) and vectors based onHSV-I (Miyanohara, A., et al., 1992, New Biologist 4:238-246; Lu, B., etal., 1995, Hepatology 21:752-759; Fong, Y., et al., 1995, Hepatology22:723-729; Tung, C., et al., 1996, Hum. Gene Ther. 7:2217-2224) havebeen evaluated for gene transfer into hepatocytes in culture and inexperimental animals. Recombinant HSV-1 vectors have been used toexpress hepatitis B virus surface antigen (HBsAG), E. coli.beta.-galactosidase, and canine factor IX-CFM in infected mouse liver(Miyanohara, A., et al., 1992, New Biologist 4:238-246). Virus stockswere either injected directly into the liver parenchyma or applied viathe portal vein. By either route, gene transfer proved to be highlyefficient and resulted in high levels of HB SAG or CFIX in thecirculation, and in a large number of .beta.-galactosidase-positivehepatocytes. Although detectable gene expression was transient, asignificant number of vector genomes was demonstrated to persist for upto two months after gene transfer. The efficiency of long term geneexpression could be increased somewhat by replacing the HCMV IE1promoter with the HSV-1 LAT promoter to direct the expression of thetransgene.

[0220] “Protein aggregation” within the scope of the present inventionincludes the phenomenon of at least two polypeptides contacting eachother in a manner that causes either one of the polypeptides to be in astate of de-solvation. This may also include a loss of the polypeptide'snative functional activity.

[0221] “De-solvation” within the scope of the present invention is astate in which the polypeptide is not in solution.

[0222] “Treating ” within the scope of the present invention reducing,inhibiting, ameliorating, or preventing. Preferably, proteinaggregation, cellular dysfunction as a result of protein aggregation andprotein-aggregation-associated diseases may be treated.

[0223] “Protein-aggregation-associated disease” within the scope of thepresent invention includes any disease, disorder, and/or affliction,protein-aggregation-associated disease include Neurodegenerativedisorders.

[0224] “Neurodegenerative disorders” are Alzheimer's disease,Parkinson's disease, prion diseases, Huntington's disease,frontotemporal dementia, and motor neuron disease. They all share aconspicuous common feature: aggregation and deposition of abnormalprotein (Table 1). Expression of mutant proteins in transgenic animalmodels recapitulates features of these diseases (A. Aguzzi and A. J.Raeber, Brain Pathol. 8, 695 (1998)). Neurons are particularlyvulnerable to the toxic effects of mutant or misfolded protein. Thecommon characteristics of these neurodegenerative disorders suggestparallel approaches to treatment, based on an understanding of thenormal cellular mechanisms for disposing of unwanted and potentiallynoxious proteins. The following is a detailed explanation of suchdiseases, their cellular malfunctions, and specific examples of theirrespective proteins that aggregate that are known thus far.

[0225] Correct folding requires proteins to assume one particularstructure from a constellation of possible but incorrect conformations.The failure of polypeptides to adopt their proper structure is a majorthreat to cell function and viability. Consequently, elaborate systemshave evolved to protect cells from the deleterious effects of misfoldedproteins. The first line of defense against misfolded protein is themolecular chaperones, which associate with nascent polypeptides as theyemerge from the ribosome, promoting correct folding and preventingharmful interactions (J. P. Taylor, et al., Science 296, 1991 (2002)).TABLE 1 Features of neurodegenerative disorders caused by proteinaggregation. Protein Toxic Disease Risk Disease deposits protein genesfactor Alzheimer's Extracellular αβ APP apoE4 disease plaques Presenilin1 allele Presenilin 2 Intracellular tau tangles Parkinson's Lewy bodiesalpha- alpha- tau disease Synuclein Synuclein linkage Parkin UCHL1 Priondisease Prion plaque PrP^(Sc) PRNP Homo- zygosity at prion codon 129Polyglutamine Nuclear and Polyglutamine- 9 different disease cytoplasmiccontaining genes with inclusions proteins CAG repeat expansion TauopathyCytoplasmic tau tau tau linkage Familial tangles SOD1 SOD1 amyotrophicBunina lateral bodies sclerosis

[0226] Alzheimer's disease is the most common neurodegenerative disease,directly affecting about 2 million Americans. It is characterized by thepresence of two lesions: the plaque, an extracellular lesion made uplargely of the β-amyloid (A) peptide, and the tangle, an intracellularlesion made up largely of the cytoskeletal protein tau. Although it ispredominantly a disease of late life, there are families in whichAlzheimer's disease is inherited as an autosomal dominant disorder ofmidlife. Three genes have been implicated in this form of the disease:the amyloid precursor protein (APP) gene (A. M. Goate, et al., Nature349, 704 (1991)), which encodes the A peptide; and the presenilinprotein genes (PS1 and PS2), which encode transmembrane proteins (R.Sherrington, et al., Nature 375, 754 (1995); E. Levy-Lahad, et al.,Science 269, 973 (1995)).

[0227] Metabolism of APP generates a variety of A species, predominantlya 40-amino acid peptide, A1-40, with a smaller amount of a 42-amino acidpeptide, A1-42. This latter form of the peptide is more prone to formingamyloid deposits. Mutations in all three pathogenic genes alter theprocessing of APP such that a more amyloidogenic species of A isproduced (D. Scheuner, et al., Nature Med. 2, 864 (1996)). Although theprecise function of the presenilins is still the subject of debate, itis clear from gene ablation experiments that presenilins are intimatelyinvolved in the COOH-terminal cleavage of A (B. De Strooper, et al.,Nature 391, 387 (1998)), and the simplest explanation of the effects ofpresenilin mutations on APP processing is that they lead to anincomplete loss of function of the complex that processes APP (L. M.Refolo, et al., J. Neurochem. 73, 2383 (1999); M. S. Wolfe et al.,Nature 398, 513 (1999)).

[0228] The implication of these findings is that the process of Adeposition is intimately connected to the initiation of Alzheimerpathogenesis and that all the other features of the disease, i.e. thetangles and the cell and synapse loss, are secondary to this initiation;this is the amyloid cascade hypothesis for Alzheimer's disease (J. A.Hardy and G. A. Higgins, Science 286, 184 (1992)). If this hypothesis iscorrect, then other genetic or environmental factors that promote Adeposition are likely to predispose to the disease, and seekingtreatments that prevent this deposition is a rational route to therapy.The only gene confirmed to confer increased risk for typical, late-onsetAlzheimer's disease is the apolipoprotein E4 allele (E. H. Corder, etal., Science 261, 921 (1993)), and apolipoprotein E gene knockouts havebeen shown to prevent A deposition (K. R. Bales, et al., Proc. Natl.Acad. Sci. U.S.A. 96, 15233 (1999)), consistent with the amyloid cascadehypothesis. Other genes predisposing to Alzheimer's disease are beingsought, and it seems most likely that they too act by alteration of Ametabolism (A. Myers, et al., Science 290, 2304 (2000); N.Ertekin-Taner, et al., Science 290, 303 (2000)).

[0229] These findings suggest that A metabolism is the key pathway to betargeted for therapy, and there has been much progress in this arenawith transgenic mice that develop plaque pathology (D. Schenk, et al.,Nature 400, 173 (1999)). Immunization of these transgenic mice with Aresults in a reduction in pathology and better performance in behavioraltests, providing evidence that A-directed therapy may be clinicallyrelevant (D. Morgan, et al., Nature 408, 982 (2000)). Immunization maynot turn out to be a practical approach to therapy, but the results ofthese animal studies have been an important proof of principle. Itshould be noted, however, that the APP transgenic mice used in thesestudies do not show tangles or cell loss, and it will be important toretest this strategy in newer, more complete models of the disease (J.Lewis, et al., Science 293, 1487 (2001)).

[0230] Parkinson's disease affects about half a million individuals inthe United States and previously has been considered a nongeneticdisorder. However, recent data increasingly implicate genetic factors inits etiology. Two genes are clearly associated with the disease:α-synuclein (PARK1) (M. H. Polymeropoulos, et al., Science 276, 2045(1997)) and parkin (PARK2) (T. Kitada, et al., Nature 392, 605 (1998)).There is evidence implicating a third, ubiquitin COOH-terminal hydrolase(PARK5) (E. Leroy, et al., Nature 395, 451 (1998); D. M. Maraganore, etal., Neurology 53, 1858 (1999)), and there are at least five otherlinkage loci (PARK 3, 4, 6, 7, and 8), indicating additionalcontributing genes (M. Farrer, et al., Hum. Mol. Genet. 8, 81 (1999); .T. Gasser, et al., Nature Genet. 18, 262 (1998); E. M. Valente, et al.,Am. J. Hum. Genet. 68, 895 (2001); C. M. Van Duijn, et al., Am. J. Hum.Genet. 69, 629 (2001); A. Hicks et al., Am. J. Hum. Genet. 69 (suppl.),200 (2001); M. Funayama, et al., Ann. Neurol. 51, 296 (2002)). Thepathological hallmark of Parkinson's disease is the deposition withindopaminergic neurons of Lewy bodies, cytoplasmic inclusions composedlargely of α-synuclein. As the work on Alzheimer's disease hassuggested, when multiple genes influence a single disorder, those genesmay define a pathogenic biochemical pathway. It is not yet clear whatthis pathway might be in Parkinson's disease. The notion that it couldbe a pathway involved in protein degradation (E. Leroy, et al., Nature395, 451 (1998)) has gained ground with the observations that parkin isa ubiquitin-protein ligase (H. Shimura, et al., Nature Genet. 25, 302(2001)) and that parkin and α-synuclein may interact (H. Shimura, etal., Science 293, 263 (2001)). In at least one patient, mutations inparkin led to Lewy body formation as seen in sporadic Parkinson'sdisease (M. Farrer, et al., Ann. Neurol. 50, 293 (2001)). Theinteraction of parkin with α-synuclein may be mediated by synphilin-1(K. K. Chung, et al., Nature Med. 7, 1144 (2001)). Anotherpathologically relevant substrate for parkin is the unfolded form ofPael, which is found to accumulate in the brains of patients with parkinmutations (Y. Imai, et al., Cell 105, 891 (2001)). If proteindegradation is the key pathogenic pathway in Parkinson's disease, onemay predict that additional Parkinson's disease loci encode otherproteins in this same pathway. Dopaminergic neurons may be moresensitive to the disease process than other neurons because they sustainmore protein damage through oxidative stress induced by dopaminemetabolism. However, work on the molecular basis of Parkinson's diseaseis currently less advanced than work on other neurodegenerativediseases; as additional genes are found, other pathogenic mechanisms mayemerge.

[0231] The most common human prion disease is sporadic Creutzfeldt-Jacobdisease (CJD). Less common are the hereditary forms, including familialCJD, Gerstmann-Straussler-Scheinker disease, and fatal familial insomnia(S. B. Prusiner, N. Engl. J. Med. 344, 1516 (2001)). Prion diseases aredistinct from other neurodegenerative disorders by virtue of theirtransmissibility. Although they share a common molecular etiology, theprion diseases vary greatly in their clinical manifestations, which mayinclude dementia, psychiatric disturbance, disordered movement, ataxia,and insomnia. The pathology of prion diseases shows varying degrees ofspongioform vacuolation, gliosis, and neuronal loss. The one consistentpathological feature of the prion diseases is the accumulation ofamyloid material that is immunopositive for prion protein (PrP), whichis encoded by a single gene on the short arm of chromosome 20.

[0232] Substantial evidence now supports the contention that prionsconsist of an abnormal isoform of PrP (J. Collinge, Annu. Rev. Neurosci.24, 519 (2001)). Structural analysis indicates that normal cellular PrP(designated PrPC) is a soluble protein rich in α-helix with littleβ-pleated sheet content. In contrast, PrP extracted from the brains ofaffected individuals (designated PrPSc) is highly aggregated anddetergent insoluble. PrPSc is less rich in helix and has a greatercontent of β-pleated sheet. The polypeptide chains for PrPC and PrPScare identical in amino acid composition, differing only in theirthree-dimensional conformation.

[0233] It is suggested that the PrP fluctuates between a native state(PrPC) and a series of additional conformations, one or a set of whichmay self-associate to produce a stable supramolecular structure composedof misfolded PrP monomers (J. Collinge, Annu. Rev. Neurosci. 24, 519(2001)). Thus, PrPSc may serve as a template that promotes theconversion of PrPC to PrPSc. Initiation of a pathogenic self-propagatingconversion reaction may be induced by exposure to a “seed” ofβ-sheet-rich PrP after prion inoculation, thus accounting fortransmissibility. The conversion reaction may also depend on anadditional, species-specific factor termed “protein X” (K. Kaneko, etal., Proc. Natl. Acad. Sci. U.S.A. 94, 10069 (1997)). Alternatively,aggregation and deposition of PrPSc may be a consequence of a rare,stochastic conformational change leading to sporadic cases. Hereditaryprion disease is likely a consequence of a pathogenic mutation thatpredisposes PrPC to the PrPSc structure.

[0234] At least nine inherited neurological disorders are caused bytrinucleotide (CAG) repeat expansion, including Huntington's disease,Kennedy's disease, dentatorubro-pallidoluysian atrophy, and six forms ofspinocerebellar ataxia (H. Y. Zoghbi and H. T. Orr, Annu. Rev. Neurosci.23, 217 (2000); K. Nakamura, et al., Hum. Mol. Genet. 10, 1441 (2001)).These are all adult-onset diseases with progressive degeneration of thenervous system that is typically fatal. The genes responsible for thesediseases appear to be functionally unrelated. The only known commonfeature is a CAG trinucleotide repeat in each gene's coding region,resulting in a polyglutamine tract in the disease protein. In the normalpopulation, the length of the polyglutamine tract is polymorphic,generally ranging from about 10 to 36 consecutive glutamine residues. Ineach of these diseases, however, expansion of the polyglutamine tractbeyond the normal range results in adult-onset, slowly progressiveneurodegeneration. Longer expansions correlate with earlier onset, moresevere disease.

[0235] These diseases likely share a common molecular pathogenesisresulting from toxicity associated with the expanded polyglutaminetract. It is now clear that expanded polyglutamine endows the diseaseproteins with a dominant gain of function that is toxic to neurons. Eachof the polyglutamine diseases is characterized by a different pattern ofneurodegeneration and thus different clinical manifestations. Theselective vulnerability of different populations of neurons in thesediseases is poorly understood but likely is related to the expressionpattern of each disease gene and the normal function and interactions ofthe disease gene product. Partial loss of function of individual diseasegenes, although not sufficient to cause disease, may contribute toselective neuronal vulnerability (I. Dragatsis, M. S. Levine, S.Zeitlin, Nature Genet. 26, 300 (2000); C. Zuccato et al. Science 293,493 (2001)). Several years ago, it was recognized that expandedpolyglutamine forms neuronal intranuclear inclusions in animal models ofthe polyglutamine diseases and the central nervous system of patientswith these diseases (C. A. Ross, Neuron 19, 1147 (1997)). Theseinclusions consist of accumulations of insoluble aggregatedpolyglutamine-containing fragments in association with other proteins.It has been proposed that proteins with long polyglutamine tractsmisfold and aggregate as antiparallel strands termed “polar zippers” (M.F. Perutz, Proc. Natl. Acad. Sci. U.S.A. 91, 5355 (1994)). Thecorrelation between the threshold polyglutamine length for aggregationin experimental systems and the CAG repeat length that leads to humandisease supports the argument that self-association or aggregation ofexpanded polyglutamine underlies the toxic gain of function. Although insome experimental systems the toxicity of expanded polyglutamine hasbeen dissociated from the formation of visible inclusions, the formationof insoluble molecular aggregates appears to be a consistent feature oftoxicity (. S. Sisodia, Cell 95, 1 (1998); I. A. Klement, et al., Cell95, 41 (1998); F. Saudou, S. Finkbeiner, D. Devys, M. E. Greenberg, Cell95, 55 (1998); P. J. Muchowski, et al., Proc. Natl. Acad. Sci. U.S.A.99, 727 (2002)). The observed correlation between aggregation andtoxicity in the polyglutamine diseases suggests a link with the otherneurodegenerative diseases characterized by deposition of abnormalprotein.

[0236] Tau has long been suspected of playing a causative role in humanneurodegenerative disease, a view supported by the observation thatfilamentous tau inclusions are the predominant neuropathological featureof a broad range of sporadic disorders, including Pick's disease,corticobasal degeneration (CBD), progressive supranuclear palsy (PSP),and the amyotrophic lateral sclerosis/parkinsonism-dementia complex.This group of disorders is collectively referred to as the “tauopathies”(V. M-Y. Lee, M. Goedert, J. Q. Trojanowski, Annu. Rev. Neurosci. 24,1121 (2001)). Filamentous tau deposition is also frequently observed inthe brains of patients with Alzheimer's disease and prion diseases. Thetau proteins are low molecular weight, microtubule-associated proteinsthat are abundant in axons of the central and peripheral nervous system.Encoded by a single gene on chromosome 17, multiple tau isoforms aregenerated by alternative splicing. The discovery that multiple mutationsin the gene encoding tau are associated with frontotemporal dementia andparkinsonism (FTDP-17) provided strong evidence that abnormal forms oftau may contribute to neurodegenerative disease (L. A. Reed, Z. K.Wszolek, M. Hutton, Neurobiol. Aging 22, 89 (2001)). Moreover,polymorphisms associated with the tau gene appear to be risk factors forsporadic CBD, PSP, and Parkinson's disease (E. R. Martin, et al., J. Am.Med. Assoc. 286, 2245 (2001); N. Cole and T. Siddique, Semin. Neurol.19, 407 (1999)). Emerging evidence suggests that tau abnormalitiesassociated with neurodegenerative disease impair tau splicing, favorfibrillization, and generally promote the deposition of tau aggregates.

[0237] Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disease of upper and lower motor neurons. About 10% ofALS cases are inherited; the remainder are believed to be sporadic cases(N. Cole and T. Siddique, Semin. Neurol. 19, 407 (1999)). Of theinherited cases, about 20% are caused by mutations in the gene encodingsuperoxide dismutase 1 (SOD1). More than 70 different pathogenic SOD1mutations have been described; all are dominant except for thesubstitution of valine for alanine at position 90, which may berecessive or dominant. Neuropathologically, ALS is characterized bydegeneration and loss of motor neurons and gliosis. Intracellularinclusions are found in degenerating neurons and glia (L. P. Rowland andN. A. Shneider, N. Engl. J. Med. 344, 1688 (2001)). Familial ALS ischaracterized neuropathologically by neuronal Lewy body-like hyalineinclusions and astrocytic hyaline inclusions composed largely of mutantSOD1.

[0238] SOD1 is a copper-dependent enzyme that catalyzes the conversionof toxic superoxide radicals to hydrogen peroxide and oxygen. Mutationsthat impair the antioxidant function of SOD1 could lead to toxicaccumulation of superoxide radicals. However, a loss-of-functionmechanism for familial ALS is unlikely given that no motor neurondegeneration is seen in transgenic mice in which SOD1 expression hasbeen eliminated. Moreover, overexpression of mutant SOD1 in transgenicmice causes motor neuron disease despite elevated SOD1 activity. Thissupports a role for a deleterious gain of function by the mutantprotein, consistent with autosomal dominant inheritance. A pro-oxidantrole for mutant SOD1 contributing to motor neuron degeneration has beenproposed. This seems unlikely, however, given that ablation of thespecific copper chaperone for SOD1, which deprives SOD1 of copper andeliminates enzymatic activity, has no effect on motor neurondegeneration in mutant SOD1 transgenic mice (J. R. Subramaniam, et al.,Nature Neurosci. 5, 301 (2002)). More recently, attention has turned tothe possible deleterious effects of accumulating aggregates of mutantSOD1. The notion that aggregation is related to pathogenesis issupported by the observation that murine models of mutant SOD1-mediateddisease feature prominent intracellular inclusions in motor neurons, andin some cases within the astrocytes surrounding them as well (D. W.Cleveland and J. Liu, Nature Med. 6, 1320 (2000)). Although a variety ofinclusions have been described in sporadic cases of ALS, there is scantevidence for deposition of SOD1 in these inclusions and no convincingevidence that aggregation contributes to the pathogenesis of sporadicALS.

[0239] It remains unclear exactly how abnormal proteins could lead toneurodegenerative disease. Determining the mechanism of toxicity ofmutant or misfolded, aggregation-prone protein remains the mostimportant unresolved research problem for each of these diseases.Although the different diseases may ultimately involve differentmechanisms, certain common themes have emerged, which could point theway to common therapeutic approaches.

[0240] Proposed mechanisms of toxicity include sequestration of criticalfactors by the abnormal protein (A. McCampbell and K. H. Fischbeck,Nature Med. 7, 528 (2001); J. S. Steffan, et al., Proc. Natl. Acad. Sci.U.S.A. 97, 6763 (2000); F. C. Nucifora, et al., Science 291, 2423(2001)), inhibition of the UPS (4), inappropriate induction of caspasesand apoptosis (M. P. Mattson, Nature Rev. Mol. Cell Biol. 1, 120(2000)), and inhibition by aggregates of neuron-specific functions suchas axonal transport and maintenance of synaptic integrity (D. W.Cleveland, Neuron 24, 515 (1999); P. F. Chapman, et al., NatureNeurosci. 2, 271 (1999)). For example, mutant polyglutamine-containingproteins bind and deplete CREB-binding protein and other proteinacetylases (A. McCampbell and K. H. Fischbeck, Nature Med. 7, 528(2001); J. S. Steffan, et al., Proc. Natl. Acad. Sci. U.S.A. 97, 6763(2000); F. C. Nucifora, et al., Science 291, 2423 (2001)). That this maycontribute to polyglutamine toxicity is supported by the finding thatdeacetylase inhibitors can mitigate the toxic effect (A. McCampbell, etal., Proc. Natl. Acad. Sci. U.S.A. 98, 15179 (2001); J. S. Steffan, etal., Nature 413, 739 (2001)). There is recent evidence that mutantpolyglutamine can impede proteasome activity (N. F. Bence, R. M. Sampat,R. R. Kopito, Science 292, 1552 (2001)); the key role of proteasomes inmaintaining cell viability indicates that this effect of the mutantprotein could be important in mediating neuronal dysfunction and death.Caspase activation and apoptosis have been well demonstrated in cellculture models of polyglutamine disease, ALS, and Alzheimer's disease(M. P. Mattson, Nature Rev. Mol. Cell Biol. 1, 120 (2000)), and the roleof apoptosis in polyglutamine disease and ALS is indicated by themitigating effects of caspase inhibition in transgenic mouse models (D.W. Cleveland, Neuron 24, 515 (1999)). Demonstration of apoptosis inpatient autopsy samples is more difficult, perhaps because of the longtime course and slow evolution of these disorders in humans or becausedifferent cell death pathways may be involved (S. Sperandio, I. deBelle, D. E. Bredesen, Proc. Natl. Acad. Sci. U.S.A. 97, 14376 (2000)).Neurofilament changes and defects in axonal transport occur in ALS (D.W. Cleveland, Neuron 24, 515 (1999)), and early synaptic pathology hasbeen found in transgenic models of Alzheimer's disease (P. F. Chapman,et al., Nature Neurosci. 2, 271 (1999)). Other implicated mechanismsinclude excitotoxicity, mitochondrial dysfunction, oxidative stress, andthe microglial inflammatory response. Indeed, downstream from the directeffects of mutant or misfolded protein in neurodegenerative diseases themechanisms of toxicity likely diverge.

[0241] These insights into the role of toxic proteins inneurodegenerative disease suggest rational approaches to treatment.First, blocking the expression or accelerating the degradation of thetoxic protein can be an effective therapy. Reducing expression of themutant polyglutamine in transgenic mice can reverse the phenotype (A.Yamamoto, J. J. Lucas, R. Hen, Cell 101, 57 (2000)), and immune-mediatedclearance of β-amyloid has a similar benefit in an animal model ofAlzheimer's disease (D. Morgan, et al., Nature 408, 982 (2000)). Becausefragments of the toxic proteins may be more pathogenic than thefull-length protein and specific cellular localization may enhancetoxicity, blocking proteolytic processing and intracellular transportare reasonable approaches to treatment. Other therapeutic strategiesinclude inhibiting the tendency of the protein to aggregate (either withitself or with other proteins), up-regulating heat shock proteins thatprotect against the toxic effects of misfolded protein, and blockingdownstream effects, such as triggers of neuronal apoptosis.Overexpression of heat shock protein can reduce the toxicity of bothmutant polyglutamine and mutant α-synuclein (J. M. Warrick, et al.,Nature Genet. 23, 425 (1999); P. K. Auluck, et al., Science 295, 865(2002)), and caspase inhibition can reduce the toxicity of bothpolyglutamine and mutant SOD (V. O. Ona, et al., Nature 399, 263 (1999);M. W. Li, et al., Science 288, 335 (2000)), indicating that therapeuticinterventions of this type may apply across multiple neurodegenerativediseases. Pharmaceutical screens are now under way to identify agentsthat block the expression or alter the processing and aggregation of thetoxic proteins responsible for neurodegenerative disease, or mitigatethe harmful effects of these proteins on neuronal function and survival.

[0242] The molecular basis for torsion dystonia remains unclear. Ozeliuset al. identified the causative gene, named TOR1A, and mapped it tohuman chromosome 9q34 (L. J. Ozelius, et al., Nature Genetics 17, 40(1997)). The TOR1A gene produces a protein named TOR-A. The majority ofpatients with early onset torsion dystonia have a unique deletion of onecodon, which results in a loss of glutamic acid (GAG) residue at thecarboxy terminal of TOR-A. A misfunctional torsin protein is produced.Notably, this was the only change observed on the disease chromosome (L.J. Ozelius, et al., Genomics 62, 377 (1999); L. J. Ozelius, et al.,Nature Genetics 17, 40 (1997)). A recent paper described an additionaldeletion of 18 base pairs or 6 amino acids at the carboxy terminus. Thisis the first mutation identified beyond the GAG deletion (L. J. Ozelius,et al., Nature Genetics 17, 40 (1997)).

[0243] In Caenorhabditis elegans, the homolog with highest amino acidsequence identity to the human TOR1A gene is the tor-2 gene product.This nematode also contains a second torsin gene named tor-1. In theoriginal paper identifying the TOR1A gene, a nematode torsin-likeprotein was described, which has since been shown to encode the ooc-5gene (L. J. Ozelius, et al., Nature Genetics 17, 40 (1997), S. E.Basham, and L. E. Rose, Dev Biol 215 253 (1999)). The three C.eleganstorsin genes share a high sequence identity to each other (L. J.Ozelius, et al., Nature Genetics 17, 40 (1997)).

[0244] The genes tor-1 and tor-2 are situated next to each other onchromosome IV of C. elegans and are oriented in the same direction.These two genes are separated by only 348 base pairs. This implies thatperhaps these genes are positioned together to form an operon unit(Blumenthal, T. 1998. Gene clusters and polycistronic transcription ineukaryotes. Bioessays 6: 480-487). Interestingly, humans also have twotorsin genes, TOR1A and TOR1B, that produce the proteins torsin A andtorsin B. These two proteins have a 70% sequence similarity (L. J.Ozelius, et al., Genomics 62, 377 (1999)). The human genes also lie onthe same chromosome (9q34), but in opposite directions (L. J. Ozelius,et al., Nature Genetics 17, 40 (1997); Ozelius L J, Hewett J W, Page CE, Bressman S B, Kramer P L, Shalish C, de Leon D, Brin M F, Raymond D,Jacoby D, Penney J, Fahn S, Gusella J F, Risch N J, Breakefield X O.1998. The gene (DYT1) for early-onset torsion dystonia encodes a novelprotein related to the Clp protease/heat shock family. Advances inNeurology. 78:93-105).

[0245] The TOR-A protein shares a distant similarity (25%-30%) to theAAA+/Hsp 100/Clp family of proteins (L. J. Ozelius, et al., Genomics 62,377 (1999); Neuwald A F, Aravind L, Spouge J L, Koonin E V. 1999. AAA+:A class of chaperone-like ATPases associated with the assembly,operation, and disassembly of protein complexes. Genome Res 9: 27-43).Members of this family are ATPases of diverse function, hinder proteinaggregation by binding to exposed surfaces, and regulate the repair ofdamaged substrates (Schirmer E C, glover J R, Singer M A, Lindquist S.1996. Hsp 100/Clp proteins: a common mechanism explains diversefunctions. Trends Biochem Sci 21:289-296) Heat shock proteins haveseveral different activities related to chaperone functions. Theyprevent misfolding of proteins, regulate protein signaling, and allowfor the correct localization of the proteins. Heat shock proteins arethought to be activated when other proteins in a cell do not foldcorrectly. If heat shock protein activation fails, misfolded proteinstend to form aggregates. This could represent a possible cause ofdiseases such as Alzheimer's, Parkinson's and Huntington's whereinprotein aggregates form.

[0246] Recently, it has been shown that the Hsp 40 and the Hsp 70 heatshock families are involved in preventing polyglutamine aggregation(Chai Y, Koppenhafer S L, Bonini N M, and Paulson H L. 1999. Analysis ofthe Role of Heat Shock Protein (Hsp) Molecular Chaperones inPolyglutamine Disease. The Journal of Neuroscience. 19(23):10338-10347)In examining the polyglutamine neurodegenerative disease spinocerebellarataxia 3, also called Machado-Joseph Disease, and its associateddisease-causing protein ataxin 3, they studied the consequences ofaggregates on the cells and the effects of chaperones on thepolyglutamine aggregates. Their experiments showed that Hsp 40 and Hsp70 are used as part of the cell's response to polyglutamine aggregates.These chaperones are able to diminish the toxic effects of theaggregates. The presence of the mutant ataxin-3 induced a stressresponse in the cells and activated the chaperone Hsp 70. Thus, the cellviews the polyglutamine protein as abnormal and recruits its chaperonesto aid in suppression of these aggregates.

[0247] Further implying that perhaps torsin proteins have a chaperonefunction was the recent finding that torsin A is localized tointracellular membranes (Kustedjo K, Bracey M H, Cravatt B F. Torsin Aand Its Torsin Dystonia-associated Mutant Forms Are LumenalGlycoproteins That Exhibit Distinct Subcellular Localizations. 2000. Jof Biol Chem 275:27933-27939). Using immunofluroescence, TOR-A was shownto have high co-localization with the ER resident protein, BiP.Interestingly, the mutant form of TOR-A, lacking a glutamic acid residueas found in dystonia patients, was located in large aggregate-likeformations absent of BiP immunoreactivity (Kustedjo K, Bracey M H,Cravatt B F. Torsin A and Its Torsin Dystonia-associated Mutant FormsAre Lumenal Glycoproteins That Exhibit Distinct SubcellularLocalizations. 2000. J of Biol Chem 275:27933-27939). This supportsanother report that torsin A is glycosylated, a characteristic of ERproteins, and is co-localized with PDI, an ER marker. Mutant TOR-A wasalso shown to develop large cytoplasmic inclusions (Hewett J,Gonzalez-Agosti C, Slater D, Ziefer P, Li S, Bergeron D, Jacoby D J,Ozelius L J, Ramesh V, and Breakefield X O. 2000. Mutant torsin A,responsible for early-onset torsion dystonia, forms membrane inclusionsin cultured neural cells. Human Molecular Genetics 9: 1403-1413).

[0248] A further embodiment of the present invention is related to ananoparticle. The polynucleotides and the polypeptides of the presentinvention may be incorporated into the nanoparticle. The nanoparticlewithin the scope of the invention is meant to include particles at thesingle molecule level as well as those aggregates of particles thatexhibit microscopic properties. Methods of using and making theabove-mentioned nanoparticle can be found in the art (U.S. Pat. Nos.6,395,253, 6,387,329, 6,383,500, 6,361,944, 6,350,515, 6,333,051,6,323,989, 6,316,029, 6,312,731, 6,306,610, 6,288,040, 6,272,262,6,268,222, 6,265,546, 6,262,129, 6,262,032, 6,248,724, 6,217,912,6,217,901, 6,217,864, 6,214,560, 6,187,559, 6,180,415, 6,159,445,6,149,868, 6,121,005, 6,086,881, 6,007,845, 6,002,817, 5,985,353,5,981,467, 5,962,566, 5,925,564, 5,904,936, 5,856,435, 5,792,751,5,789,375, 5,770,580, 5,756,264, 5,705,585, 5,702,727, and 5,686,113).

[0249] A further embodiment of the present invention is related tomicrorarrays. The polynucleotides and the polypeptides of the presentinvention may be incorporated into the microarrays. The microarraywithin the scope of the invention is meant to include particles at thesingle molecule level as well as those aggregates of particles thatexhibit microscopic properties. Methods of using and making theabove-mentioned nanoparticle can be found in the art (U.S. Pat. No.6,004,755)

[0250] The present invention is explained in more detail with the aid ofthe following embodiment examples.

EXAMPLES Methods and Materials

[0251] Plasmid Constructs

[0252] The tor-2 cDNA was isolated from whole worm mRNA using RT-PCRwith the following primers. Primer 1(5′-AACGCGTCGACAATGAAAAAGTTCGCTGAAAAATGGTTTCTATTG 3′) (SEQ ID NO. 11)and primer 2 (5′ AAGGCCTTCACAACTCATCATTAAACTCTTTCTTCG) (SEQ ID NO. 12).Briefly, total RNA was isolated from a mixed population of C. elegansusing TriReagent (Molecular Research Center) followed by mRNA isolationusing the PolyATtract mRNA Isolation System III (Promega) and cDNAsynthesis using the Superscript First-Strand Synthesis System for RT-PCRfrom Life Technologies. Confirmation of the predicted ORF (WormBaseY37AlB.13) was performed by sequencing. Mutant versions of the tor-2cDNA were generated using PCR-mediated site-directed mutagenesis. Toobtain the Δ368 mutant form of tor-2 an initial round of PCR wasperformed to generate an approximately 1 kb cDNA (corresponding to aminoacids 1-367) using primer 1 and primer 3 (5′GGGAAAAATTCAAGATCAAGAACTCTTTGCATG 3′) (SEQ ID NO. 13). In parallel, anapproximately 200 bp fragment (corresponding to amino acids 369-412) wasamplified with primer 2 and primer 4 (5′CATGCAAAGAGTTCTTGATCTTGAATTTTTCCC) (SEQ ID NO. 14). The two fragmentswere then combined and amplified using primers 1 and 2 to reconstructthe complete cDNA. The ΔNDEL form of tor-2 was also generated using PCRwith the following primers. Primer 5 (5′CTAGCTAGCATGAAAAAGTTCGCTGAAAAATGG 3′) (SEQ ID NO. 15) and primer 6,which lacks DNA encoding the terminal NDEL amino acids (SEQ ID NO. 16)(5′ GGGGTACCTCAAAACTCTTTCTTCGAATTGAGTG 3′) (SEQ ID NO. 17) wereutilized. Mutant forms of tor-2 were confirmed by sequencing. All tor-2cDNAs were subcloned into vector pPD30.38 using the enzymes Nhe I andKpn I (Fire, A, Harrison, S W, Dixon, D. 1990. A modular set of lacZfusion vectors for studying gene expression in Caenorhabditis elegans.Gene 93:189-198.).

[0253] The plasmids unc-54::Q19-GFP and unc-54::Q82-GFP were provided asa generous gift from Dr. Rick Morimoto, Northwestern University (Satyal,S, Schmidt, E, Kitagaya, K, Sondheimer, N, Lindquist, S T, Kramer, J,Morimoto, R. 2000). Polyglutamine aggregates alter protein foldinghomeostasis in Caenorhabditis elegans. Proc Natl Acad Sci USA97:5750-5755.).

[0254]C. elegans Protocols

[0255] Nematodes were maintained using standard procedures (Brenner, S.1974. The genetics of Caenorhabditis elegans. Genetics. 77:71-94). Amixture of the plasmids encoding the polyglutatmine-GFP fusions andtorsin constructs were co-injected with the rol-6 marker gene into thegonads of early-adult hermaphrodites. The injection mixtures containedpPD30.38-Q82-GFP or pPD30.38-Q19-GFP, pRF4 (the rol-6[su1006] dominantmarker) using standard microinjection procedures, and eitherpPD30.38-tor-2, pPD30.38-Δ368 tor-2, or pPD30.38-ΔNDELtor-2 (Mello C C,Kramer J M, Stinchcomb D, Ambros V. 1991. Efficient gene transfer in C.elegans: Extrachromosomal maintenance and integration of transformingsequences. EMBO J 10: 3959-3970 1992). For each combination of plasmidDNAs, worm lines expressing the extrachromosomal arrays were obtained.Following stable transmission of the arrays, integration into the genomewas performed using gamma irradiation with 3500-4000 rads from a Cobalt60 (Inoue, T, Thomas, J. 2000. Targets of TGF-signaling inCaenorhabditis elegans dauer formation. Develop. Biol. 217:192-204).Stable integrated lines were obtained for all constructs.

[0256] Fluorescence Microscopy

[0257] Worms were examined using a Nikon Eclipse E800 epifluorescencemicroscope equipped with an Endow GFP HYQ and Texas Red HYQ filter cubes(Chroma, Inc.). Images were captured with a Spot RT CCD camera(Diagnostic Instruments, Inc.). MetaMorph Software (Universal Imaging,Inc.) was used for pseuodocoloration of images, image overlays, andaggregate size quantitation. Statistical analysis of aggregate size andquantity was performed using the software Statistica.

RESULTS

[0258] Isolation of a cDNA Encoding C. elegans TOR-2 and Site-DirectedMutagenesis

[0259] As an important resource for several lines of experimentation, acDNA corresponding to the full-coding region predicted for the C.elegans tor-2 gene was isolated. The predicted open-reading frame wasconfirmed and found to be completely correct by DNA sequencing of bothstrands. All exon and intron boundaries were confirmed as well. This wasimportant because the TOR-2 protein encoded by this gene contains aunique N-terminal portion not found in the other torsins of C elegans(FIGS. 1-3). The 1.3 kb tor-2 cDNA encodes a predicted protein of 412amino acids. A single protein from the cDNA of the approximately correctmolecular weight (49 Kd) is recognized in C. elegans extracts by TOR-2specific peptide antisera. The tor-2 cDNA was subcloned into thepPD30.38 vector under the control of the C. elegans unc-54 promoterelement which is expressed in body wall muscle cells (Fire, A, Harrison,S W, Dixon, D. 1990. A modular set of lacZ fusion vectors for studyinggene expression in Caenorhabditis elegans. Gene 93:189-198; Satyal, S,Schmidt, E, Kitagaya, K, Sondheimer, N, Lindquist, S T, Kramer, J,Morimoto, R. 2000). Two modifications of the tor-2 cDNA were alsogenerated for initial structure-function analysis of the TOR-2 protein.Both of these modified cDNAs were subcloned into pPD30.38. Usingsite-directed mutagenesis, a cDNA designed to mimic the expression ofthe dominant negative protein that causes primary torsion dystonia inhumans was created (Ozelius L J, Hewett J W, Page C E, Bressman S B,Kramer P L, Shalish C, de Leon D, Brin M F, Raymond D, Corey D P, FahnS, Risch N J, Buckler A J, Gusella J F, Breakefield X O. 1997. Theearly-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein.Nature Genetics 17: 40-48.). This consisted of a mutant tor-2 cDNAlacking a codon at amino acid 368, which encodes serine. In humans, thecorresponding amino acid deletion in TOR1A is glutamic acid. Both serineand glutamic acid are polar amino acids. Additionally, a tor-2 cDNA witha deletion of the four most C-terminal amino acids (NDEL) in the TOR-2protein was produced. The NDEL sequence is a putative ER-retentionsignal (data not shown).

[0260] Co-Expression of TOR-2 Suppresses Polyglutamine Repeat-InducedProtein Aggregation

[0261] Satyal and coworkers (2000) have created artificial aggregates ofpolyglutamine-repeats fused to GFP that are ectopically expressed in thebody wall muscle cells of C. elegans using the well characterized unc-54promoter. Aggregation of the GFP reporter protein is dependent on thelength of the polyglutamine tract. For example, body wall expression ofa fusion of 19 glutamines (Q19) to GFP does not reflect a change innormally cytoplasmic, evenly distributed, and diffuse GFP localization(FIG. 4a). However, a tract of 82 glutamines (Q82) fused to GFP resultsin a distinct change in GFP localization wherein discrete aggregates areclearly evident in all animals (FIG. 4b).

[0262] Following introduction of the appropriate vector (unc-54::tor-2cDNA) and selection of stable transgenic animals, co-expression of theTOR-2 protein under the control of the same high-level constitutivepromoter dramatically reduces both the number of GFP-containingaggregates in animals containing Q82-GFP (FIG. 4c). In fact, diffusebody wall muscle fluorescence reappears in many of these animals aswell. Co-expression of TOR-2 with Q19 does not alter the normal,cytoplasmic distribution of GFP and thus does not appear to induceaggregation. In contrast, co-expression Q82-GFP with TOR-2 containingthe site-directed deletion of amino acid 368 (Δ368) in the C-terminus ofthis protein is not capable of restoring the body wall fluorescence inthese animals (FIG. 4d). Interestingly, co-expression of TOR-2 Δ368 withQ19 does not change the general cytoplasmic localization of GFP fromwhat is found in Q19-GFP animals.

[0263] There is a statistically significant difference in the size ofQ82-GFP aggregates among the various constructs. The average size ofaggregates from thirty each of Q82, Q82+TOR-2, and Q82+TOR-2 Δ368animals was recorded. The average size of aggregates from Q82 animalswas 2.7 μm compared with 1.6 μm from Q82+TOR-2 (FIG. 5). This differenceis significant (p<0.001) using a pair-wise t-test. Furthermore, thedifference in aggregate size between Q82 and Q82+TOR-2 Δ368 animals wasalso significant (p<0.001) with an aggregate size of 4.8 μm forQ82+TOR-2 Δ368 animals (compared with 2.7 μm for Q82). These differencesare easily observed with photomicrographs, as shown in FIG. 6a-6 b.

[0264] Additionally, the amount of variability in aggregate size differsamong the transgenic constructs. When aggregate size is classified intothe following categories, 0-3 μm, 3-5 μm, 5-9 μm, and 9-26 μm,aggregates from Q82 animals display a 63%, 25%, 9%, and 3% distribution,respectively (Table 2). Animals co-expressing Q82 and TOR-2 demonstratefar less variability in aggregate size with 90% of the aggregates in thesmallest size group and only 7% and 3% of the aggregates in the 3-5 μmand 5-9 μm categories, respectively. Conversely, the aggregates fromanimals co-expressing Q82 and TOR-2 Δ368 demonstrate a large degree ofvariability with 16% aggregates in both the 5-9 μm, and 9-26 μmcategories. TABLE 2 Variability of Q82 Aggregate Size aggregates weregrouped according to size for each different treatment. Percentages werecalculated based on the total number of aggregates for each treatment.Size of Aggregate Q82 + (μm) Q82 Q82 + TOR-2 TOR-2/^(Δ)368 0 to 3 63%90% 48% 3 to 5 25%  7% 20% 5 to 9  9%  3% 16%  9 to 26  3% 16%

[0265] There is a generalized growth defect associated with the Q82-GFPstrain. This strain exhibits a reduced growth rate (as judged by larvalstaging at specific time points) in comparison to wild-type animals(Satyal, S, Schmidt, E, Kitagaya, K, Sondheimer, N, Lindquist, S T,Kramer, J, Morimoto, R. 2000. Polyglutamine aggregates alter proteinfolding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci USA97:5750-5755). Both wild-type and mutant torsin were co-expressed withQ82-GFP in order to determine if the torsin protein alleviated thisapparent homeostatic burden (FIGS. 4a-4 d). Co-expression with wild-typetor-2 had no obvious effect on the growth inhibition associated withQ82-GFP animals. However, tor-2 Δ368 co-expression significantlyexacerbated the growth inhibitory effect such that 71% of the animalswere still at the L1/L2 stage of development compared with 46% ofQ82-GFP animals 48 hours after parental egg laying. Neither tor-2 Δ368co-expression with Q19 nor wild-type tor-2 changed the growth rate ofanimals (See Table 3). TABLE 3 Growth Analysis Adults were allowed tolay eggs for a set length of time and then removed from plate. Offspringwere counted 48 hours after parental removal according to larval stage.L1/L2 L3 L4/Adult Total N2 2 (0.5%) 78 (20%) 309 (79%) 389 Q19 2 (14%)184 (63%) 68 (23%) 292 Q82 134 (46%) 149 (51%) 7 (3%) 290 Q19/tor-2 99(18%) 395 (73%) 46 (9%) 540 Q82/tor-2 122 (42%) 140 (48%) 27 (10%) 289Q19/Δ368 44 (19.2%) 159 (69.4%) 26 (11.4%) 229 Q82/Δ368 98 (71%) 40(29%) 1 (0.007%) 139

[0266] Co-Expression of other Torsin Genes Suppresses PolyglutamineRepeat-Induced Protein Aggregation.

[0267] Experiments were perform in accordance with the above-describedQ82+tor-2 coexpression experiments except that tor-2 was replaced withooc-5 and TOR-A, i.e. Q82+ooc-5 and Q82+TOR-A experiments. Further, Q82was coexpressed with ooc-5 and tor-2 (i.e. Q82+tor-2+ooc-5). FIGS.10c-10 e demonstrate that, like tor-2 alone, expression of ooc-5, TOR-A,and tor-2+ooc-5, respectively, with Q82 resulted in a more diffusepattern of Q82 expression and a reduction of Q82 aggregates. Further,expression of TOR-2 in combination with OOC-5 results in an apparentenhanced reduction in the size of the Q82 aggregates. Perhaps, this isan indication that such torsin proteins are present at least in part ina complex.

[0268] Polyglutamine Aggregate Accumulation Over Time

[0269] Q19-GFP animals had tiny aggregates when they reached adulthoodand the aggregates increased in size as the animals aged. Specifically,adult worms expressing Q19-GFP, Q19-GFP+TOR-2, or Q19-GFP+TOR-2 Δ368were analyzed each day for seven days and aggregate size scored (FIG.7). Worms expressing Q19-GFP had an average aggregate size of 7.5 μm onday 1 of adulthood and 7.9 μm on day 2. The size of the aggregatesincreased to 8.9 μm on day 3 and decreased on day 4 to 8.5 μm. Theaverage size fluctuated slightly on days 5, 6 and 7, but stayed close toan average size of 8.2 μm. Worms co-expressing TOR-2 were found to havesignificantly smaller aggregates. On day 1, the average size of theaggregates was 4.8 μm. The size of the aggregates decreased andstabilized over time with an average size of 3.0 μm on day 4 and anaverage size of 3.8 μm on day 6. Notably, aggregates from wormsco-injected with TOR-2 Δ368 continued to increase in size each day. Onthe first day the average aggregate size was 10.3 μm; by day 4 it was12.8 μm and on the last day of analysis the aggregates averaged 15.0 μmin size. Statistical analysis revealed no significant difference overtime. However, there was a difference in the results of treatment andthese differences persisted over time. Those with TOR-2 proteintreatment had smaller aggregate size on average (3.9 μm) and wereconsistently smaller when compared with aggregate size for Q82, whichwas 8.2 μm on average. Mutant torsin protein averaged 12.8 μm and wassignificantly different from both wild-type torsin protein and Q82.

[0270] TOR-2 Antibody and SDS-PAGE

[0271] A SDS-PAGE of whole worm protein extracts and subsequent westernblot were performed and the blot stained with TOR-2 antibody (FIG. 8).It showed the level of TOR-2 protein to be minimal in wild-type N2worms, Q19 and Q82 worms. TOR-2 protein levels of Q19/TOR-2, Q82/TOR-2,Q19+TOR-2/368 and Q82+TOR-2/Δ368 revealed higher levels than N2, Q19,and Q82. However, the levels among the 4 constructs of wild-type andmutant torsin were equivalent. Actin controls were used and weredetermined to be equivalent for all worms used.

[0272] Antibody Staining

[0273] Whole worms stained with TOR-2 antibody showed diffuse stainingthroughout the worm (FIG. 9). However, distinctly higher levels oftorsin localization were seen in a tight ring completely surrounding theaggregates in the Q82 worms.

Discussion

[0274] Early-onset torsion dystonia is caused by a dominant mutationresulting in the loss of a glutamic acid residue at the carboxy terminusof TOR-A. The majority of dystonia cases exhibit this deletion; thisindicates that this region is critical for correct functioning of theprotein. It was recently shown that members of the AAA+ family form asix-member oligomeric ring. This ring structure is used in theassociations with other proteins. Ozelius et al., (1997) hypothesizedthat this area of the glutamic acid deletion could be a criticalcomponent of the ring structure, if TOR-A forms a ring. The loss of thisamino acid could affect the relationship of TOR-A with surroundingproteins (Ozelius L J, Hewett J W, Page C E, Bressman S B, Kramer P L,Shalish C, de Leon D, Brin M F, Raymond D, Corey D P, Fahn S, Risch N J,Buckler A J, Gusella J F, Breakefield X O. 1997. The early-onset torsiondystonia gene (DYT1) encodes an ATP-binding protein. Nature Genetics 17:40-48).

[0275] An in vivo assay was utilized to examine the effects of torsinson polyglutamine aggregates. Co-expression of the TOR-2 proteins withQ82 reduced the formation of the aggregates in body-wall muscle cells.Antibody localization studies of Q82+TOR-2 revealed that the TOR-2protein appeared to be surrounding the aggregate in a tight,doughnut-like fashion. This is interesting as it gave us the firstindication of how these proteins could be interacting with theaggregates.

[0276] Formation of aggregates and their presence in intracellularinclusions is a hallmark of many neurodegenerative diseases. All cellshave a system to deal with misfolded or damaged proteins. This system iscalled the ubiquitin-proteasome pathway (UPS). This system works by“tagging” the protein to be degraded with ubiquitin. Therefore, theprotein becomes a target for degradation. However, recent reportsindicate that this pathway is hindered by the presence of proteinaggregates (Bence et al., 2001). By expressing two proteins known toinduce the formation of aggregates, Bence et al., were able tocompletely restrain the UPS. This led to a buildup of proteins taggedwith ubiquitin that the cells were not able to remove. This build-up,plus additional misfolded proteins, led to cell death (Bence N F, SampatR M, Kopito R R. Impairment of the Ubiquitin-Proteasome System byProtein Aggregation. Science 292:1552-1555).

[0277] Johnston et al. (1998), described a different structure from theproteasome system called the aggresome (Johnston J A, Ward C L, Kopito RR. Aggresomes: A Cellular Response to Misfolded Proteins. 1998. J ofCell Biology 143(7): 1883-1898). In a related review by Kopito et al.(2000), they describe the cell's inability to remove aggregated proteinsas “cellular indigestion” (Kopito R R, Sitia R. Aggresomes and RussellBodies. 2000. EMBO Reports 1(3): 225-231). Their theory is thataggresomes are a response to this “cellular indigestion.” When thecell's ability to destroy protein aggregates is surpassed, the aggresomeis formed. The formation of the aggresome is a result of cell stress. Itis highly organized structurally. However, aggresomes are only formed atthe microtubule organizing center (MTOC). Microtubules (MT) are used totransport the aggregated or misfolded proteins to the aggresome fordegradation. Intermediate filaments are also required and are rearrangedin a specific manner in order to form a supporting framework for theaggresome. Aggresomes contain high amounts of proteasomes fordegradation, ubiquitin, and molecular chaperones. Interestingly,inclusions, which are found in many neurodegenerative disorders, alsocontain varying amounts of the same components as found in aggresomes.These inclusions contain the disease-causing protein aggregates.Therefore, there is a clear link between “cellular indigestion” anddisease (Johnston J A, Ward C L, Kopito R R. Aggresomes: A CellularResponse to Misfolded Proteins. 1998. J of Cell Biology 143(7):1883-1898; Kopito R R, Sitia R. Aggresomes and Russell Bodies. 2000.EMBO Reports 1(3): 225-231).

[0278] Based on the antibody localization and the fact that TOR-2 isable to reduce the aggregates and restore partial body-wall staining, itis interesting to speculate that perhaps TOR-2 is involved in theubiquitin-proteasome pathway and/or in ER-associated degradation.Co-expression of the mutant tor-2, TOR-2/Δ368, with Q82 is not able torestore partial diffuse body wall staining as seen with wild-type TOR-2and actually seemed to worsen the aggregates. This supports the theorythat this portion of the gene is essential for correct functioning.Deletion of the NDEL region of tor-2, which bears homology to the ERlocalization signal, KDEL, did not exacerbate the aggregates as seenwith the TOR-2/Δ368 (data not shown). With the deletion of the NDEL,TOR-2 is presumably not retained in the ER and is presumably free in thecytoplasm. Perhaps, it is at a higher concentration and is able tointeract better with the aggregates. Also, the growth analysis datasuggests that the “glutamic acid region” is critical for growth as 71%of these worms remained at L1/L2 stages 48 hours after egg-layingcompared with 46% of the Q82 worms.

[0279] The data support a role for TOR-2 as a molecular chaperone.Further, the data support that TOR-A, and ooc-5 are molecular chaperonesas well. This is the first clear demonstration that at least oneactivity of torsin proteins is chaperone activity. Further, these torsinproteins clearly reduce the amount of Q82 protein aggregation in vivo.

[0280] TOR-A is co-localized with α-synuclein in Lewy bodies ofParkinson's patients. Alpha-synuclein is misfolded in these inclusions.Torsins could help proteins fold correctly or assist in the degradationof misfolded proteins via the ubiquitin-proteasome system. The fact thatthe antibody localization shows the torsin protein as a tight ringaround the aggregate suggests more of a degradative role. It was able torestore partial body wall staining when co-expressed with Q82, whichmeans that the aggregates were removed. Although aggregates were stillpresent, they were smaller when compared with Q82 alone.

[0281] The Q19 age analysis study showed that aggregates worsen overtime. This is true with many diseases, such as Huntington's patients, inwhich the patients deteriorate as time progresses. This model could haveimplications for drug therapies. TOR-2 is able to reduce the aggregates.This model also showed that TOR-2 was able to keep the size of theaggregates at a baseline and stable level, while the aggregatesco-expressed with TOR-2/Δ368 grew larger over time. Hopefully, TOR-2could be used as a therapeutic agent. While it may not completelyalleviate the symptoms completely, it could keep the patient's conditionat a stable level instead of deteriorating as time progresses. Perhapsan enhanced effect could be observed with the co-expression of TOR-1, asthese may function in a complex.

[0282] The data, combined with the aggresome theory, suggests that manydiseases, such as dystonia, are the result of the cell's inability tocope with the aggregated proteins. These protein aggregates affect otherproteins and could, in fact, cause a cascade-like effect. This isthought to be the mechanism behind prion diseases, such as spongioformencephalopathy. The fact that the aggregate size of TOR-2 Δ368+Q82 islarger when compared with Q82 alone suggests that the mutant version mayserve as a starting point for other proteins to misfold and formaggregates. TOR-2 appears to play a multi-dimensional role in the celland is widely expressed.

[0283] Numerous modifications and variations on the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

[0284] All of the references, as well as their cited references, citedherein are hereby incorporated by reference with respect to relativeportions related to the subject matter of the present invention and allof its embodiments.

What is claimed is:
 1. An isolated polynucleotide, comprising SEQ ID NO.1 or SEQ ID NO.
 3. 2. A vector, comprising the isolated polynucleotideaccording to claim
 1. 3. A host cell, comprising the isolatedpolynucleotide according to claim
 1. 4. A method for making a torsinpolypeptide, comprising culturing the host cell according to claim 3 fora duration of time under conditions suitable for expression of torsinpolypeptide.
 5. A composition, comprising the polynucleotide accordingto claim 1 and at least one physiologically-acceptable carrier.
 6. Amicroarray, comprising the polynucleotide according to claim
 1. 7. Ananoparticle, comprising the polynucleotide according to claim
 1. 8. Atransgenic animal, comprising the polynucleotide according to claim 1.9. An isolated polynucleotide, comprising a nucleic acid sequence thatis at least 90% identical to the polynucleotide according to claim 1.10. An isolated polynucleotide, comprising a nucleic acid sequence thatis at least 80% identical to the polynucleotide according to claim 1.11. An isolated polynucleotide, comprising a nucleic acid sequence thatis at least 70% identical to the polynucleotide according to claim 1.12. A vector, comprising the isolated polynucleotide according to claim11.
 13. A host cell, comprising the isolated polynucleotide according toclaim
 11. 14. A method for making a torsin polypeptide, comprisingculturing the host cell according to claim 13 for a duration of timeunder conditions suitable for expression of torsin polypeptide.
 15. Acomposition, comprising the polynucleotide according to claim 11 and atleast one physiologically-acceptable carrier.
 16. A microarray,comprising the polynucleotide according to claim
 11. 17. A nanoparticle,comprising the polynucleotide according to claim
 11. 18. A transgenicanimal, comprising the polynucleotide according to claim
 11. 19. Anisolated polynucleotide, which hybridizes at 65° C. in the presence of abuffer comprising 0.1×SSC and 0.1% SDS toat least 15 consecutivenucleotides of the isolated polynucleotide according to claim 11 and hastorsin activity or at least 15 nucleotides of a complement thereof. 20.A process for detecting polynucleotide sequences which encode apolypeptide with at least 70% homology to a polypeptide having an aminoacid sequence of SEQ ID NO. 2 or SEQ ID NO. 4 and having torsinactivity, comprising (a) hybridizing the isolated polynucleotideaccording to claim 11; (b) expressing the polynucleotide to produce apolypeptide; (c) detecting the presence or absence of torsin activity ofthe polypeptide.
 21. A method for detecting a polynucleotide thatencodes a polypeptide having torsin activity, comprising contacting apolynucleotide sample with at least 15 consecutive nucleotides of thepolynucleotide according to claim 11 and having torsin activity, or atleast 15 consecutive nucleotides of a complement thereof.
 22. A methodfor producing a polynucleotide encoding a polypeptide having torsinactivity, comprising contacting a polynucleotide sample with apolynucleotide comprising at least 15 consecutive nucleotides of thepolynucleotide according to claim 11 and having torsin activity, or atleast 15 consecutive nucleotides of the complement thereof.
 23. A methodof reducing protein aggregation in vivo or in vitro, comprisingadministering the polynucleotide according to claim 11 to a human beingor an animal in need thereof.
 24. A method of treating at least oneprotein-aggregation-associated disease comprising administering thepolynucleotide according to claim 11 to a human being or an animal inneed thereof.
 25. The method according to claim 24, wherein the at leastone protein-aggregation-associated disease is selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, Prion disease,Polyglutamine disease, Tauopathy, Huntington's disease, Dystonia, andFamilial amyotrophic lateral sclerosis.
 26. A method of treatingsymptoms of at least one protein-aggregation-associated diseasecomprising administering the polynucleotide according to claim 11 to ahuman being or an animal in need thereof.
 27. The method according toclaim 26, wherein the at least one protein-aggregation-associateddisease is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Prion disease, Polyglutamine disease, Tauopathy,Huntington's disease, Dystonia, and Familial amyotrophic lateralsclerosis.
 28. An isolated polynucleotide, which encodes a polypeptidehaving an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO.
 4. 29. Avector, comprising the isolated polynucleotide according to claim 28.30. A host cell, comprising the isolated polynucleotide according toclaim
 28. 31. A method for making a torsin polypeptide, comprisingculturing the host cell according to claim 30 for a duration of timeunder conditions suitable for expression of torsin polypeptide.
 32. Acomposition, comprising the polynucleotide according to claim 28 and atleast one physiologically-acceptable carrier.
 33. A microarray,comprising the polynucleotide according to claim
 28. 34. A nanoparticle,comprising the polynucleotide according to claim
 28. 35. A transgenicanimal, comprising the polynucleotide according to claim
 28. 36. Anisolated polypeptide, comprising SEQ ID NO. 2 or SEQ ID NO.
 4. 37. Anisolated antibody, wherein said antibody binds the isolated polypeptideaccording to claim
 36. 38. An isolated polypeptide, comprising an aminoacid sequence that is at least 90% identical to the polypeptideaccording to claim
 36. 39. A transgenic animal, comprising the isolatedpolypeptide according to claim
 36. 40. A composition, comprising theisolated polypeptide according to claim 38 and at least onephysiologically-acceptable carrier.
 41. A microarray, comprising theisolated polypeptide according to claim
 36. 42. A nanoparticle,comprising the isolated polypeptide according to claim
 36. 43. Anisolated polypeptide, comprising an amino acid sequence that is at least80% identical to the polypeptide according to claim
 36. 44. An isolatedpolypeptide, comprising an amino acid sequence that is at least 70%identical to the polypeptide according to claim
 36. 45. A transgenicanimal, comprising the isolated polypeptide according to claim
 44. 46. Acomposition, comprising the isolated polypeptide according to claim 44and at least one physiologically-acceptable carrier.
 47. A microarray,comprising the isolated polypeptide according to claim
 44. 48. Ananoparticle, comprising the isolated polypeptide according to claim 44.49. An isolated antibody, wherein said antibody binds the isolatedpolypeptide according to claim
 44. 50. A method of reducing proteinaggregation comprising administering the isolated polypeptide accordingto claim 44 to a human being or an animal in need thereof.
 51. A methodof treating at least one protein-aggregation-associated disease,comprising administering the isolated polypeptide according to claim 44to a human being or an animal in need thereof.
 52. The method accordingto claim 51, wherein the at least one protein-aggregation-associateddisease is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Prion disease, Polyglutamine disease, Tauopathy,Huntington's disease, Dystonia, and Familial amyotrophic lateralsclerosis.
 53. A method of treating symptoms of at least oneprotein-aggregation-associated disease comprising administering theisolated polypeptide according to claim 44 to a human being or an animalin need thereof.
 54. The method according to claim 53, wherein the atleast one protein-aggregation-associated disease is selected from thegroup consisting of Alzheimer's disease, Parkinson's disease, Priondisease, Polyglutamine disease, Tauopathy, Huntington's disease,Dystonia, and Familial amyotrophic lateral sclerosis.
 55. A method ofcontrolling the expression of at least one isolated polypeptide havingan amino acid sequence that is at least 70% identical to SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, or SEQ ID NO. 10 in anorganism, comprising administrating at least one an polynucleotidehaving a nucleic acid sequence that is at least 70% identical to SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, or SEQ ID NO. 9 to theorganism.
 56. The method according to claim 55, wherein the at least oneisolated polypeptide is administered to C. elegans.
 57. A method ofreducing protein aggregation comprising administering an isolatedpolypeptide comprising an amino acid sequence that is at least 70%identical SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, or SEQID NO. 10 to a human being or an animal in need thereof.
 58. A method oftreating at least one protein-aggregation-associated disease comprisingadministering an isolated polypeptide comprising an amino acid sequencethat is at least 70% identical SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,SEQ ID NO. 8, or SEQ ID NO. 10 to a human being or an animal in needthereof.
 59. The method according to claim 58, wherein the at least oneprotein-aggregation-associated disease is selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, Prion disease,Polyglutamine disease, Tauopathy, Huntington's disease, Dystonia, andFamilial amyotrophic lateral sclerosis.
 60. A method of treating thesymptoms of at least one protein-aggregation-associated diseasecomprising administering an isolated polypeptide comprising an aminoacid sequence that is at least 70% identical SEQ ID NO. 2, SEQ ID NO. 4,SEQ ID NO. 6, SEQ ID NO. 8, or SEQ ID NO. 10 to a human being or ananimal in need thereof.
 61. The method according to claim 60, whereinthe at least one protein-aggregation-associated disease is selected fromthe group consisting of Alzheimer's disease, Parkinson's disease, Priondisease, Polyglutamine disease, Tauopathy, Huntington's disease,Dystonia, and Familial amyotrophic lateral sclerosis.